Negotiation in China Research Paper Example | Topics and Well Written Essays - 1250 words

Negotiation in China - Research Paper Example   Many entrepreneurs have strived to make their ways in China with different investment intentions that include creating a partnership with Chinese firms. Negotiations are never easy since there are special insights that are required for proper negotiations processes. There exist a number of complications that hinder negotiations inChinaa which are largely based on the complicated Chinese culture. Better negotiation styles and skills are therefore very necessary in bringing competitive advantages and benefits. Chinese culture has very great influence on her business negotiations. China’s culture and behavior should be viewed differently by investors since the economics within Greater China become more internally linked together. To create competitive advantage among foreign investors going to china, there is a dire need to train them in international manners and customs so that they can learn a better negotiation style that is appropriate for China. The collectivism, hierarc hical, relationship and the haggling culture of Chinese people should be well understood particularly their long courting, formal and the fact that they prefer drawing on intermediaries when it comes to negotiation process. Dr. Robert Grosse authorized this research to further investigate the business managers’ perceptions regarding specific characteristics needed for successful negotiation in China. Dr. Robert B. Rogow, Dean of the College of Business and Technology at Eastern Kentucky University, will receive the finalized report on March 6, 2013.

Ibn Ishaqs Life of Muhammad Essay Example | Topics and Well Written Essays - 500 words

Ibn Ishaqs Life of Muhammad - Essay Example One of the rulers continued in power â€Å"†¦until God sent Muhammad the prophet. I was told on the authority of al-Zuhri that he said Chosroes wrote to Badhan the following letter: ‘I have been told that a man of the Quraysh has come forth in Mecca asserting that he is a prophet.’†1 Muhammad grew up in the â€Å"brackish settlement† of Mecca, as Muhammad ibn Abd Allah.2 His father, allegedly, died before he was born. Then, to make matters worse, when Muhammad was only six years old, his mother died, leaving him an orphan. He became a shepherd and worked for his uncle. This left Muhammad in a difficult place until he became a camel driver for caravans up until about age 40. He also struggled with poverty for much of this time. Muhammad’s life was to end in triumph, however—even though it had begun with unusual sorrow. Muhammad received a vision from an angel giving him the dictum of Islam, and Ibn Ishaq recounts Muhammad’s feelings as he prepared to leave for what is now called Medina from his home city of Mecca (this journey would be called hijira). It was â€Å"†¦unusual for Ibn Ishaq to give such an intimate account of Muhammad’s state of mind [where he was praying].

Toxicity and Autoactivation of Baits Experiment

Toxicity and Autoactivation of Baits Experiment Abstract Alternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results. 1. Introduction 1.1. Nervous System The human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001; Purves et al, 2004 and Thompson,1993). 1.2. Cells of CNS The brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporting cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types: astrocytes, oligodentrocytes and the microglial cells. 1.2.1. Glial Cells Astrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003; Gee et al, 2004 and Longuemare et al, 1999). Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999; Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory re sponses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has paved path for finding targets and therapeutics for many diseases.(Bhatia, 2008; Huang et al 2008; Hwang et al, 2008 and Kim et al, 2008). 1.2.2. Neurons Neuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project. The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites. All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma throughout the axon known as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Pu rves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001). Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the Patch clamp method have made the study of these ion channels easier (Bear et al, 2001). 1.3. Ion channel Ion channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004). Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, ÃŽ ³-aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004). 1.4. Voltage Gated Calcium Channel (VGCC) Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005). The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under â€Å"calcium channelopathies† (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to act as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their bin ding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000; Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997). 1.5. Calcium channel structure A calcium channel consists of five important subunits ÃŽ ±1. ÃŽ ±2, ÃŽ ², ÃŽ ´ and ÃŽ ³. The ÃŽ ±1 subunit is known as the pore forming complex (Yang et al, 2006). The ÃŽ ±1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of ÃŽ ±1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006). The intracellular loop of the ÃŽ ±1 subunit has interaction site for the binding ÃŽ ² subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of ÃŽ ² subunit to the tryptophan residue is important for controlling the gating of ÃŽ ±1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the ÃŽ ±1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or c lose the pore (Horn et al, 2000). The ÃŽ ² subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, ÃŽ ²1, ÃŽ ²2, ÃŽ ²3 and ÃŽ ²4. Different isoforms of the ÃŽ ² subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with ÃŽ ± subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The ÃŽ ±2 subunit is also known as the ÃŽ ±2/ÃŽ ´ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The ÃŽ ±2 and ÃŽ ´ subunits are linked together by disulphide bonds. Like other subunits ÃŽ ±2/ÃŽ ´ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin is a drug known to act on ÃŽ ±2/ÃŽ ´ subunit, but their binding affinity varies with different isoforms of the ÃŽ ´ subunit (Luo et al, 2001 and Luo et al, 2002).T he ÃŽ ³ subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006). The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006). 1.6. Calcium Channel Types Calcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes. Voltage Dependent Inactivation (VDI) responsible for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thoug ht to depend on the ÃŽ ² subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006). 1.7. Calcium Channel Gene The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different ÃŽ ±1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the CavÃŽ ±1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006). The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6). Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of ÃŽ ±1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002). 1.8. Spino Cerebellar Ataxia 6 Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The ÃŽ ±1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggr egate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest in the C-terminal of the ÃŽ ±1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing. 1.9. Splicing Transcription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006; Saba et al 2005; Auboeuf et al, 2007; Jurica et al, 2003; Hicks et al, 2005 and Manley et al, 2006). In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008). Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of â€Å"RNA world†. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999; Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004; Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005). Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004; Raymond et al, 2004; Sarao et al, 1991 and Schaller et al, 1992). 1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005). The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004; Gray et al, 2007; Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005). Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998). These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6. 1.11. Protein protein interaction studies Protein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002). 1.12. Yeast Two Hybrid System (Y2H) Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our ÃŽ ²-galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999). The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their dis advantages (Criekinge et al, 1999 and Luban et al, 1995) The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995) The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999). Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation. 2. Aim This study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein. Baits 5inSER is a 472 base pair length protein with ggcag NLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008). The aims of the project are To test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment. 3. Materials and Methods 3.1. Control Mating 3.1.1. Control strains The control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait [pGBKT7-53] and Y187 with the target [pGADT7-T] , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187[pGADT7-T] The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures. 3.1.2. Small Scale Yeast Transformation A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30à ¢Ã‚ Ã‚ ° C. The overnight culture was diluted in 50 ml of YPAD to an OD600 Toxicity and Autoactivation of Baits Experiment Toxicity and Autoactivation of Baits Experiment Abstract Alternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results. 1. Introduction 1.1. Nervous System The human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001; Purves et al, 2004 and Thompson,1993). 1.2. Cells of CNS The brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporting cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types: astrocytes, oligodentrocytes and the microglial cells. 1.2.1. Glial Cells Astrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003; Gee et al, 2004 and Longuemare et al, 1999). Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999; Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory re sponses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has paved path for finding targets and therapeutics for many diseases.(Bhatia, 2008; Huang et al 2008; Hwang et al, 2008 and Kim et al, 2008). 1.2.2. Neurons Neuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project. The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites. All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma throughout the axon known as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Pu rves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001). Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the Patch clamp method have made the study of these ion channels easier (Bear et al, 2001). 1.3. Ion channel Ion channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004). Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, ÃŽ ³-aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004). 1.4. Voltage Gated Calcium Channel (VGCC) Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005). The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under â€Å"calcium channelopathies† (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to act as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their bin ding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000; Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997). 1.5. Calcium channel structure A calcium channel consists of five important subunits ÃŽ ±1. ÃŽ ±2, ÃŽ ², ÃŽ ´ and ÃŽ ³. The ÃŽ ±1 subunit is known as the pore forming complex (Yang et al, 2006). The ÃŽ ±1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of ÃŽ ±1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006). The intracellular loop of the ÃŽ ±1 subunit has interaction site for the binding ÃŽ ² subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of ÃŽ ² subunit to the tryptophan residue is important for controlling the gating of ÃŽ ±1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the ÃŽ ±1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or c lose the pore (Horn et al, 2000). The ÃŽ ² subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, ÃŽ ²1, ÃŽ ²2, ÃŽ ²3 and ÃŽ ²4. Different isoforms of the ÃŽ ² subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with ÃŽ ± subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The ÃŽ ±2 subunit is also known as the ÃŽ ±2/ÃŽ ´ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The ÃŽ ±2 and ÃŽ ´ subunits are linked together by disulphide bonds. Like other subunits ÃŽ ±2/ÃŽ ´ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin is a drug known to act on ÃŽ ±2/ÃŽ ´ subunit, but their binding affinity varies with different isoforms of the ÃŽ ´ subunit (Luo et al, 2001 and Luo et al, 2002).T he ÃŽ ³ subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006). The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006). 1.6. Calcium Channel Types Calcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes. Voltage Dependent Inactivation (VDI) responsible for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thoug ht to depend on the ÃŽ ² subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006). 1.7. Calcium Channel Gene The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different ÃŽ ±1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the CavÃŽ ±1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006). The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6). Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of ÃŽ ±1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002). 1.8. Spino Cerebellar Ataxia 6 Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The ÃŽ ±1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggr egate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest in the C-terminal of the ÃŽ ±1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing. 1.9. Splicing Transcription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006; Saba et al 2005; Auboeuf et al, 2007; Jurica et al, 2003; Hicks et al, 2005 and Manley et al, 2006). In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008). Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of â€Å"RNA world†. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999; Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004; Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005). Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004; Raymond et al, 2004; Sarao et al, 1991 and Schaller et al, 1992). 1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005). The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004; Gray et al, 2007; Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005). Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998). These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6. 1.11. Protein protein interaction studies Protein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002). 1.12. Yeast Two Hybrid System (Y2H) Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our ÃŽ ²-galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999). The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their dis advantages (Criekinge et al, 1999 and Luban et al, 1995) The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995) The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999). Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation. 2. Aim This study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein. Baits 5inSER is a 472 base pair length protein with ggcag NLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008). The aims of the project are To test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment. 3. Materials and Methods 3.1. Control Mating 3.1.1. Control strains The control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait [pGBKT7-53] and Y187 with the target [pGADT7-T] , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187[pGADT7-T] The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures. 3.1.2. Small Scale Yeast Transformation A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30à ¢Ã‚ Ã‚ ° C. The overnight culture was diluted in 50 ml of YPAD to an OD600

The Last Full Measure :: essays research papers

The Last Full Measure is a vividly detailed account of the events that took place in the Civil War after the Battle of Gettysburg. In the novel, the author tells the story of the war after July of 1863 from several points of view. He uses three main characters to depict these points: Ulysses S. Grant, General of the U.S. Army, Robert E. Lee, General of the Confederate Army, and Joshua L. Chamberlain, a simple professor from Maine. The reader can gain a detailed understanding of the war by seeing it through Grant and Lee’s eyes. The reader can experience a more firsthand account of war by seeing it from Chamberlain’s point of view, who is not a professional solder. After reading this novel, the reader begins to realize what war actually means. To Lee, it is fighting for a way of life, while for Grant, it is the value of the Union and it’s dwindling future. The reader also realizes the hardships that come from war. For Lee, it would be not being able to see his family grow, while for Grant, it would be his struggle with alcoholism and depression. Also while reading the book, the reader is able to pick up two sides to almost every situation, the sides of Lee and Grant. In the foreword, Shaara states that his objective is to tell the reader the feelings of the men of the Civil War, which he achieves by using an immense collection of knowledge to probe into the lives of the soldiers. Shaara gives the audience an objective novel that shows the hardships in one of the greatest wars ever fought. Toward the end, Shaara takes a clear, anti-war stand. It is obvious that he realizes why wars are fought, but cannot understand why we have never learned from our mistakes. The novel is ended eloquently by showing the war’s effects on each of the main characters and what has become of them in the time after the war. Lee tries to make up lost time with his family, while Grant serves two terms as President and later enjoys retirement. Chamberlain was awarded the Congressional Medal of Honor and later became a governor. Both Lee and Grant eventually publish their memoirs at the end of the novel and look back on the war, which is told by Shaara to be a key event in The Last Full

Dr. Seuss’s -Too Many Daves Essay

The search for personal identity is said to be a lifetime endeavor. At a certain point of their lives, most people eventually recognize who they truly are. Others live all their lives in confusion or fear of confronting their real selves because these might be something they would not like. Some people search long and hard to discover their essential Self. Others grow up truthful to themselves so that they develop an identity early in their lives and thus are able to develop more fully throughout their lifetime. Some people, meanwhile—the confused and afraid—construct an image that they use to deceive others and even themselves. Identity, therefore, is an issue that normal individuals grapple with. It is not only a grown-up issue, too. Even children have to deal with understanding who and what they are. They face the issue of a more simplistic but no less relevant nature than adults do on the subject. Dr. Seuss’s children’s poetry â€Å"Too Many Daves† can be interpreted as a piece that deals with the subject of personal identity and individuality, discussing it on a level that a young child could understand and find enjoyable and interesting at the same time. The persona in the narrative verse is a disinterested third-person speaker who simply would like to tell a story that he thinks others would be interested to know. The characters in his story is Mrs. McCave and her twenty-three sons, all named Dave. Maybe Mrs. McCave thought that it would be easier to simply choose one name for all her sons rather than think of a unique one each time she bears another son. She had twenty-three of them, after all. The disadvantage of this, she finds out later on, is that when she calls one son, all twenty-three Daves would come to answer her call. Ironically, she only realizes this when all of them are grown-ups already and it has become too late to rename them. Furthermore, now that they are all grown-ups, she is now able to think of unique and creative names by which she could call each one. The most obvious and catching attribute of the poem, even before its funny story, is its lilting cadence. All lines uniformly begin with an iamb followed by three anapests. This particular pattern may have been used by Dr. Seuss not so much in consonance with the meaning of the poem but because of its musical effect. When a storyteller or anyone reading-aloud this particular story begins with the first line â€Å"Did I ever tell you? † (1), the upward inflection on the second and fourth words, he would certainly pique the interest of any child-listener. The succeeding lines proceed with a gallop-like speed and sound so that the even if the listener cannot follow the story behind the lines or is simply too young to understand, he would enjoy the rhythm of the reading—as long as the reader places the proper emphases in the proper places, of course. Most Dr. Seuss books are notable for their nonsensical words that appeal not so much for the content or subject matter of the verse but for the effect of the words on speech when the verses are read or on the ear when they are listened to. â€Å"Too Many Daves† is no exception to this Dr. Seuss trademark. The regular rhythm of the verses and the inclusion of words which are purely Dr. Seuss’s inventions and, therefore, not meant to be understood, gives the piece a tone of playfulness prompts the reader to treat the story lightheartedly. The charm of Dr. Seuss’s stories is how they allow the reader to explore beyond the common and everyday things, whether he was conscious about this or not while he was writing them. â€Å"Too Many Daves† has a story which sounds too incredible to be true, but for its young listeners or readers, children whose ability to accept the fantastic and impossible are not yet hindered by the imagination-constricting ability to rationalize which adults are unfortunate to have developed along with growing-up. Aside from the rhythm, the element of heavy musicality is also affected by the profusion of rhyme, alliteration and assonance within the twenty-four lines. The reason that the mother-character is named Mrs. McCave is so that it would rhyme with the son’s name, Dave. The entire poem is composed of couplet rhymes with a different rhyme for every couplet. Alliteration is evident in lines like â€Å"twenty-three† (2), â€Å"she wishes that when they were born†(9) and in some of the names the mother enumerates for her sons like â€Å"Stuffy† and â€Å"Stinky† (14), â€Å"Ziggy† and â€Å"Soggy Muff† (17), â€Å"Buffalo Bill† and Biffalo Buff† (18) and â€Å"Weepy Weed† (19). Assonance, meanwhile, is present in almost all lines starting with the â€Å"a† sound in â€Å"that wasn’t a smart thing† (3) to â€Å"Yoo-Hoo† (4), â€Å"come on the run† (6), and the names â€Å"Hoos-Foos† (11), â€Å"Hot-Shot† (12), â€Å"Marvin O’Gravel Balloon Face† (16), â€Å"Soggy Muff† (17), â€Å"Sneepy† and â€Å"Weepy Weed† (19), â€Å"Oliver Boliver Butt† (23) and â€Å"Zanzibar Buck-Buck McFate† (23). The enumeration of possible names that the mother wishes she has named for her sons is the most interesting aspect of the poem because of the creativity that must have gone in the writer’s mind in thinking of aurally appealing as well as thought-provoking names for the McCave boys—although Dr. Seuss might have intentionally meant to make them nonsensical and meaningless when he wrote them. The reader of â€Å"Too Many Daves† can assume, however, that the mother must have been inspired to think of these names in particular because they represent the personalities of her children, which she only sees and observes as each child grows up. Every reader can employ his own creativity as he imagines the metaphorical or literal meanings behind every name. Perhaps Bodkin Van Horn is the son who dresses sharply. Hoos-foos sounds like someone who does not take things in life seriously. Simms is the average guy who is liked by everyone. Hot-Shot is an athletic jock. Sunny Jim is the optimistic one. Shadrack is a homebody. Blinkey has something wrong with his eyes. Stuffy is always serious and uptight. Stinkey does not like to take regular baths. Putt-putt is cute but childish. Moon Face is overweight and loves to eat. Marvin O’Gravel Balloon Face is like his brother, Moon Face, but more lazy. Ziggy is nice and ordinary. Soggy Muff is untidy. Buffalo Bill always figures in a fight. Biffalo Buff is always safe and stays away from troubles. Sneepy is sickly and weak. Weepy Weed is a crybaby. Paris Garters dresses well. Harris Tweed, too. Sir Michael Carmichael Zutt thinks too highly of himself. Oliver Boliver Butt is a simpleton while Zanzibar Buck-Muck McFate hates the small town life and dreams of being an explorer or an astronaut someday. The final line of the poem summarizes the message that the speaker would like to emphasize regarding the weird situation of the McCave boys’ being singularly named: â€Å"But she didn’t do it. And now it’s too late. (24). One’s name is the first step by which the individual defines his identity. Personality would eventually emerge whatever one’s name is, but it would have helped the McCave brothers if they were given unique names by their mother, and hence unique identities in her regard of each son. By depriving them of individual names, it appears like she had liked her sons to be the same in every aspect. It could also be interpreted to mean that she does not care much about his sons as individuals with their respective identities and personalities. She just realizes too late that even if they are all her sons she could not control the nature of humans to grow differently from each other. On the other hand, however, one could argue that the mother did not name her children with various names because she does not want them to be burdened by the expectations that some names may have on their owners. For instance, if a person were named Lovely at birth, it would be unfortunate if she grows up be unattractive. Mrs. McCave would like his sons to develop their own identities and personalities and later on, decide to get a name appropriate to them. Work Cited Dr. Seuss. Too Many Daves.

High Performance Working Systems Essay

High performance working systems have been defined as a combination of the practices of HR, the work structure of the organization and the processes utilized by the organization in order to maximize the knowledge, skills, commitment and flexibility of the employees. When this is implemented effectively, this form of working system would be able to benefit both the employees and management of the organization since employees will become more involved in the organization by becoming valuable contributors to the overall growth of the company (Bohlander & Snell, 2007). In the case of Lanier Worldwide, the high performance working system would benefit the company since there are a number of branches that are situated around the country, utilizing a high performance working system would ensure that they are given up-to-date information of the company’s business plans and strategies which would allow the employees from the different branches to provide suggestions in order to improve the operations of the company as well as making the employees more committed to the courses of action being implemented by the company (Bohlander & Snell, 2007). While all of the steps in setting up a high performance working system are considered to be crucial, the most important would have to be the establishment of a solid two-way communication plan during the initial stages of the changes being made by the company using this form of working system. This is because when management is able to properly communicate with their employees, fears and concern that may be felt by employees will be diminished. This, in turn, will ensure that commitment towards the established goals of the company will be reaffirmed and allow issues and areas of improvement to easily be addressed. Without a properly crafted communication plan, employees may become apathetic to the changes management would want to implement which may open doors to more problems on the part of the company’s operation as a whole.