Enzyme Kinetics

Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes, with a focus on their reaction rates. The study of an enzyme's kinetics reveals the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or a poison might inhibit the enzyme.

Enzymes are usually protein molecules that manipulate other molecules — the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzymatic mechanism. These mechanisms can be divided into single-substrate and multiple-substrate mechanisms. Kinetic studies on enzymes that only bind one substrate, such as triosephosphate isomerase, aim to measure the affinity with which the enzyme binds this substrate and the turnover rate.

Flagellum Motar

The flagellum is linked to a flexible hook. The hook is attached to a series of protein rings , which are embedded in the outer and inner (plasma) membranes. The rings form a rotor, which rotates with the flagellum at more than 100 revolutions per second. The rotation is driven by a flow of protons through an outer ring of proteins , the stator, which also contains the proteins responsible for switching the direction of rotation

LTP Mechanisms

Long-term potentiation (LTP) is the long-lasting improvement in communication between two neurons that results from stimulating them simultaneously. Since neurons communicate via chemical synapses, and because memories are believed to be stored within these synapses, LTP is widely considered one of the major cellular mechanisms that underlies learning and memory.

LTP shares many features with long-term memory that make it an attractive candidate for a cellular mechanism of learning. For example, LTP and long-term memory are triggered rapidly, each depends upon the synthesis of new proteins, each has properties of associativity, and each can potentially last for many months. LTP may account for many types of learning, from the relatively simple classical conditioning present in all animals, to the more complex, higher-level cognition observed in humans.

By enhancing synaptic transmission, LTP improves the ability of two neurons, one presynaptic and the other postsynaptic, to communicate with one another across a synapse. The precise mechanisms for this enhancement of transmission have not been fully established, in part because LTP is governed by multiple mechanisms that vary by such things as brain region, animal age, and species. Yet in the most well understood form of LTP, enhanced communication is predominantly carried out by improving the postsynaptic cell's sensitivity to signals received from the presynaptic cell. These signals, in the form of neurotransmitter molecules, are received by neurotransmitter receptors present on the surface of the postsynaptic cell. LTP improves the postsynaptic cell's sensitivity to neurotransmitter in large part by increasing the activity of existing receptors and by increasing the number of receptors on the postsynaptic cell surface.

LTP was discovered in the rabbit hippocampus by Terje Lømo in 1966 and has remained a popular subject of research since. Most modern LTP studies seek to better understand its basic biology, while others aim to draw a causal link between LTP and behavioral learning. Still others try to develop methods, pharmacologic or otherwise, of enhancing LTP to improve learning and memory. LTP is also a subject of clinical research, for example, in the areas of Alzheimer's disease and addiction medicine.


Long-term potentiation occurs through a variety of mechanisms throughout the nervous system; no single mechanism unites all of LTP's many types. However, for the purposes of study, LTP is commonly divided into three phases that occur sequentially: short-term potentiation, early LTP, and late LTP. Little is known about the mechanisms of short-term potentiation,

Each phase of LTP is governed by a set of mediators, small molecules that dictate the events of that phase. These molecules include protein receptors that respond to events outside of the cell, enzymes that carry out chemical reactions within the cell, and signaling molecules that allow the progression from one phase to the next. In addition to these mediators, there are also modulator molecules, described later, that interact with mediators to finely alter the LTP ultimately generated.

The early (E-LTP) and late (L-LTP) phases of LTP are each characterized by a series of three events: induction, maintenance, and expression. Induction is the process by which a short-lived signal triggers that phase of LTP to begin. Maintenance corresponds to the persistent biochemical changes that occur in response to the induction of that phase. Expression entails the long-lasting cellular changes that result from activation of the maintenance signal. Thus the mechanisms of LTP can be discussed in terms of the mediators that underlie the induction, maintenance, and expression of E-LTP and L-LTP.

Adult Stem Cells Used To Rebuild Heart Tissue

Genome editing with engineered nucleases

Genome editing with engineered nucleases (GEEN) refers to a reverse genetics method using ‘molecular scissors’, or artificially engineered nucleases, to cut and create specific double-stranded break (DSB) at desired locations in the genome, harnessing the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ). There are currently 3 families of engineered nucleases being used: Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALENs), and engineered meganuclease re-engineered homing endonucleases.
It is commonly practiced in genetic analysis that in order to understand the function of a gene or a protein function one interferes with it in a sequence-specific way and monitors its effects on the organism. However, in some organisms it is difficult or impossible to perform site-specific mutagenesis, and therefore more indirect methods have to be used, such as silencing the gene of interest by short RNA interference (siRNA) . Yet gene disruption by siRNA can be variable and incomplete. Genome editing with nucleases such as ZFN is different from siRNA in that the engineered nuclease is able to modify DNA-binding specificity and therefore can in principle cut any targeted position in the genome, and introduce modification of the endogenous sequences for genes that are impossible to specifically target by conventional RNAi. Furthermore, the specificity of ZFNs and TALENs are enhanced as two ZFNs are required in the recognition of their portion of the target and subsequently direct to the neighboring sequences. It was chosen by Nature Methods as the 2011 Method of the Year.
Modern biology has greatly benefited from reverse genetics: the ability to start from particular genotypes and then look at the resultant phenotypes. Given that phenotypic changes are often complex and a result of multiple genetic interactions reverse genetics has been particularly significant in modern biology because of its inherent simpler nature. Among the key aspects of reverse genetic analysis is the ability to modify the genetic code. Although techniques such as site-directed mutagenesis allow such studies more in vitro settings, in vivo observations of phenotypic effects of the genetic changes provide a more comprehensive view of mutational significance. This has been made possible in yeast and mice by recombination based methods, which use the recombination machinery of cells to exchange DNA between a naturally occurring gene in the organism and an exogenous DNA source with the desired characteristics. However such techniques are less successful in other organisms and additionally require stringent selection steps and thus addition of selection specific sequences to the incorporated into the DNA which are a deviation from the naturally occurring genetic sequences. Furthermore they can be quite inefficient as only 1 of a million mouse embryonic stem cells treated with donor DNA incorporated it at the target sequence. Use of other techniques such as P-element transgenesis in Drosophila also have their limitations, the major one being the randomness of incorporation and the possibility of affecting other genes and expression patterns. However, genomic editing with engineered nucleases is a rapidly growing technology with the promise of overcoming these shortcomings by the use of relatively simple concepts.
Double stranded breaks and their repair
First and foremost in understanding the use of nucleases in genome editing is the understanding of DNA double stranded break (DSB) repair mechanisms. Two of the known DSB repair pathways that are essentially functional in all organisms are the non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ uses a variety of enzymes to directly join the DNA ends in a DSB while in HDR, a homologous sequence is utilized as a template for regeneration of missing DNA sequence at the break point. The natural properties of these pathways form the very basis of nucleases based genome editing. NHEJ is error prone such that it was shown to cause mutations at the repair site in approximately 50% of DSB in mycobacteria and also its low fidelity has been linked to mutational accumulation in leukemias. Thus if one is able to create a DSB at a desired gene in multiple samples, it is very likely that mutations will be generated at that site in some of the treatments because of errors created by the NHEJ infidelity. On the other hand, the dependency of HDR on a homologous sequence to repair DSBs can be exploited by inserting a desired sequence within a sequence that is homologous to the flanking sequences of a DSB which, when used as a template by HDR system, would lead to the creation of the desired change within the genomic region of interest. Despite the distinct mechanisms, the concept of the HDR based gene editing is in a way similar to that of homologous recombination based gene targeting. However, the rate of recombination is increased by at least three orders of magnitude when DSBs are created and HDR is at work thus making the HDR based recombination much more efficient and eliminating the need for stringent positive and negative selection steps. So based on these principles if one is able to create a DSB at a specific location within the genome, then the cell’s own repair systems will help in creating the desired mutations.
Site-specific double stranded breaks
Creation of a DSB in DNA should not be a challenging task as the commonly used restriction enzymes are capable of doing so. However, if genomic DNA is treated with a particular restriction endonuclease many DSBs will be created. This is a result of the fact that most restriction enzymes recognize a few base pairs on the DNA as their target and very likely that particular base pair combination will be found in many locations across the genome. To overcome this challenge and create site-specific DSB, three distinct classes of nucleases have been discovered and bioengineered to date. These are the Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and meganucleases. Here we provide a brief overview and comparison of these enzymes and the concept behind their development.
Current engineered nucleases
Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific. This can be exploited to make site-specific DSB in genome editing; however, the challenge is that not enough meganucleases are known, or may ever be known, to cover all possible target sequences. To overcome this challenge, mutagnesis and high throughput screening methods have been used to create megnuclease variants that recognize unique sequences. Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence. Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucelases in a method named rationally designed meganuclease (US Patent 8,021,867 B2). Meganuclease have the benefit of causing less toxicity in cells compared to methods such as ZFNs likely because of more stringent DNA sequence recognition; however, the construction of sequence specific enzymes for all possible sequences is costly and time consuming as one is not benefitting from combinatorial possibilities that methods such as ZFNs and TALENs utilize. So there are both advantages and disadvantages.
As opposed to meganucleases, the concept behind ZFNs and TALENs is more based on a non-specific DNA cutting enzyme which would then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs). The key to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not common among restriction enzymes. Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity. A restriction enzyme with such properties is FokI. Additionally FokI has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner would recognize a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases would avoid the possibility of unwanted homodimer activity and thus increase specificity of the DSB. Although the nuclease portion of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically happen in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins such as transcription factors. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Zinc fingers have been more established in these terms and approaches such as modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries among other methods have been used to make site specific nucleases.
Over the past decade, efficient genome editing has been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and the method holds a promising future in becoming a standard experimental strategy in research labs. The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants testifies to the significance of the methods and the list is expanding rapidly. Genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove genomic elements and therefore create complex systems. In addition, gene functions can be studied using stem cells with engineered nucleases. Listed below are some specific tasks this method can carry out:
  • Targeted gene mutation 
  • Creating chromosome rearrangement 
  • Study gene function with stem cells 
  • Transgenic animals
  •  Endogenous gene labeling 
  • Targeted transgene addition
  Targeted gene addition in plants
Genome editing using ZFN provides a new strategy for genetic manipulation in plants and is likely to assist engineering desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes.Progress in such cases have been recently reported in Arabidopsis thaliana and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations. In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case. Such genome modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation. Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting. These include the reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of introduction or induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.
Gene therapy
The ideal gene therapy practice is that which replaces the defective gene with a normal allele at its natural location. This is advantageous over a virally delivered gene as there is no need to include the full coding sequences and regulatory sequences when only a small proportions of the gene needs to be altered as is often the case. The expression of the partially replaced genes is also more consistent with normal cell biology than full genes that are carried by viral vectors. ZFN-induced targeting can also attack defective genes at their endogenous chromosomal locations. Examples include the treatment of X-linked severe combined immunodeficiency (X-SCID) by ex vivo gene correction with DNA carrying the interleukin-2 receptor common gamma chain (IL-2Rγ) with the correct sequence. Insertional mutagenesis by the retroviral vector genome induced leukemia in some patients, a problem predicted to be avoided by GEEN and ZFNs. However, ZFNs may also cause off-target mutations, in a different way from viral transductions. Currently many measures are taken to improve off-target detection and ensure safety before treatment. Recently, Sangamo BioSciences (SGMO) introduced the Delta 32 mutation (a suppressor of CCR5 gene which is a co-receptor for HIV-1 entry into T cells therefore enabling HIV infection) using Zinc Finger Nuclease (ZFN). Their results were presented at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) held in Chicago from September 17–20, 2011. Researchers at SGMO mutated CCR5 in CD4+ T cells and subsequently produced an HIV-resistant T-cell population.
Reference: Genome editing with engineered nucleases. (2012, June 13). In Wikipedia, The Free Encyclopedia. Retrieved 10:59, July 23, 2012, from

Penicillin Binding Protein Animation

Penicillin-binding proteins (PBPs) are a group of proteins that are characterized by their affinity for and binding of penicillin. They are a normal constitutent of many bacteria;All beta-lactam antibiotics bind to PBP to have their effect of preventing cell wall construction by the bacterium.

There are a large number of PBPs, usually several in each organism, and they are found as both membrane-bound and cytoplasmic proteins. For example, Spratt (1977) reports that six different PBPs are routinely detected in all strains of E. coli ranging in molecular weight from 40000 to 91000. The different PBPs occur in different numbers per cell and have varied affinities for penicillin . The PBPs are usually broadly classified into high-molecular-weight (HMW) and low-molecular-weight (LMW) categories.

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PBPs are all involved in the final stages of the synthesis of peptidoglycan, which is the major component of bacterial cell walls. Bacterial cell wall synthesis is essential to growth, cell division (thus reproduction) and maintaining the cellular structure in bacteria. Inhibition of PBPs leads to irregularities in cell wall structure such as elongation, lesions, loss of selective permeability, and eventual cell death and lysis.
PBPs have been shown to catalyse a number of reactions involved in the process of synthesising cross-linked peptidoglycan from lipid intermediates and mediating the removal of D-alanine from the precursor of peptidoglycan. Purified enzymes have been shown to catalyse the following reactions: D-alanine carboxypeptidase, peptidoglycan transpeptidase, and peptidoglycan endopeptidase. In all bacteria that have been studied, enzymes have been shown to catalyse more than one of the above reactions (Spratt, 1977). The enzyme has a penicillin-insensitive transglycosylase N-terminal domain (involved in formation of linear glycan strands) and a penicillin-sensitive transpeptidase C-terminal domain (involved in cross-linking of the peptide subunits) and the serine at the active site is conserved in all members of the PBP family
PBPs bind β-lactam antibiotics because they are similar in chemical structure to the modular pieces that form the peptidoglycan (Disteche and Bouille et al, 1982). When they bind to penicillin, the β-lactam amide bond is ruptured to form a covalent bond with the serine residue at the PBPs active site. This is an irreversible reaction and inactivates the enzyme.
There has been a great deal of research into PBPs because of their role in antibiotics and resistance. Bacterial cell wall synthesis and the role of PBPs in its synthesis is a very good target for drugs of selective toxicity because the metabolic pathways and enzymes are unique to bacteria (Chambers, 1999). Resistance to antibiotics has come about through overproduction of PBPs and formation of PBPs that have low affinity for penicillins (among other mechanisms such as lactamase production). Research on PBPs has led to the discovery our new semi-synthetic β-lactams, wherein altering the side-chains on the original penicillin molecule has increased the affinity of PBPs for penicillin, and, thus, increased effectiveness in bacteria with developing resistance.
The β-lactam ring is an analogous structure to all β-lactam antibiotics. Shown here is the core structure of penicillin, the square in the middle with an oxygen double-bonded to it is the β-lactam ring

CCR5 Antagonist

CCR5 receptor antagonists are a class of small molecules that antagonize the CCR5 receptor. The C-C motif chemokine receptor CCR5 is involved in the process by which HIV, the virus that causes AIDS, enters cells. Hence antagonists of this receptor are entry inhibitors and have potential therapeutic applications in the treatment of HIV infections.The life cycle of the HIV presents potential targets for drug therapy, one of them being the viral entry pathway. The C-C motif chemokine receptors CCR5 and CXCR4 are the main chemokine receptors involved in the HIV entry process. These receptors belong to the seven transmembrane G-protein-coupled receptor (GPCR) family and are predominantly expressed on human T-cells, dendritic cells and macrophages, Langerhans cells.They play an important role as co-receptors that HIV type 1 (HIV-1) uses to attach to cells before viral fusion and entry into host cells.[1] HIV isolates can be divided into R5 and X4 strains. R5 strain is when the virus uses the co-receptor CCR5 and X4 strain is when it uses CXCR4. The location of CCR5 receptors at the cell surface, both large and small molecules have the potential to interfere with the CCR5-viral interaction and inhibit viral entry into human cells.

Mechanism of action HIV enters host cells in the blood by attaching itself to receptors on the surface of the CD4+ cell.[8] Viral entry to the CD4+ cell begins with attachment of the R5 HIV-1 glycoprotein 120 (gp120) to the CD4+ T-cell receptor, which produces a conformational change in gp120 and allows it to bind to CCR5, thereby triggering glycoprotein 41 (gp41) mediated fusion of the viral envelope with the cell membrane and the nucleocapsid enters the host cell. CCR5 co-receptor antagonists prevent HIV-1 from entering and infecting immune cells by blocking CCR5 cell-surface receptor. Small molecule antagonists of CCR5 bind to a hydrophobic pocket formed by the transmembrane helices of the CCR5 receptor. They are thought to interact with the receptor in an allosteric manner locking the receptor in a conformation that prohibits its co-receptor function.

Truvada - How it works against HIV

Tenofovir/emtricitabine, trademark Truvada, is a fixed-dose combination of two antiretroviral drugs used for the treatment of HIV. It consists of 300 milligrams of tenofovir and 200 milligrams of emtricitabine. By combining the two agents into one tablet, it reduces the pill burden and increases compliance with antiretroviral therapy. The drug has been examined for use as a pre-exposure prophylaxis against HIV infection. A Cochrane review found that both tenofovir alone, as well as the tenofovir/emtricitabine combination, significantly decreased the risk of contracting HIV. The Food and Drug Administration approved it for prophylactic use on July 16, 2012. The drug has side effects including: nausea, vomiting, dizziness, loss of appetite and diarrhea, liver and kidney toxicity and loss of bone density.
The HEAT study (randomized, double-blind, placebo-matched, multicentre) showed that once-daily emtricitabine/tenofovir plus lopinavir/ritonavir or boosted atazanavir or efavirenz were effective in the initial treatment of patients with HIV-1 infection (with screening plasma HIV-1 RNA levels of ≥1,000,000 copies/mL in ACTG 5202). In other randomized trials, emtricitabine/tenofovir DF 200 mg/300 mg once daily was an effective backbone for boosted protease inhibitor (PI)-based regimens in the initial treatment of HIV-1 infection. Emtricitabine/tenofovir DF in combination with various boosted PIs was generally well tolerated by adults with HIV-1 infection. Truvada was developed by Gilead Sciences and approved by the United States Food and Drug Administration in 2004. A combination pill containing Truvada and efavirenz (Sustiva) is also available and is marketed as Atripla.

Stem Cells Animation

Stem cells are primal cells common to all multi-cellular organisms that retain the ability to renew themselves through cell division and can differentiate into a wide range of specialized cell types. Research in the human stem cell field grew out of findings by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s

The three broad categories of mammalian stem cells are: embryonic stem cells, derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells are able to differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells.

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As stem cells can be readily grown and transformed into specialised cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture, their use in medical therapies has been proposed. In particular, embryonic cell lines, autologous embryonic stem cells generated through therapeutic cloning, and highly plastic adult stem cells from the umbilical cord blood or bone marrow are touted as promising candidates.

Defining properties

The rigorous definition of a stem cell requires that it possesses two properties:
Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
Unlimited potency - the capacity to differentiate into any mature cell type. In a strict sense, this makes stem cells either totipotent or pluripotent, although some multipotent and/or unipotent progenitor cells are sometimes referred to as stem cells.

These properties can be illustrated in vitro, using methods such as clonogenic assays, where the progeny of single cell is characterized. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.

Potency definitions

Potency specifies the differentiation potential of the stem cell.

, embryonic stem cells originate as inner mass cells with in a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.

Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types.

Multipotent stem cells can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.).

Unipotent cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells.

Embryonic stem cells
Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst. A blastocyst is an early stage embryo - approximately 4 to 5 days old in humans and consisting of 50-150 cells. ES cells are pluripotent, and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

Embryonic stem cell

When given no stimuli for differentiation, ES cells will continue to divide in vitro and each daughter cell will remain pluripotent. The pluripotency of ES cells has been rigorously demonstrated in vitro and in vivo, thus they can be indeed classified as stem cells.

Because of their unique combined abilities of unlimited expansion and pluripotency, embryonic stem cells are a potential source for regenerative medicine and tissue replacement after injury or disease. To date, no approved medical treatments have been derived from embryonic stem cell research. This is not surprising considering that many nations currently have moratoria on either ES cell research or the production of new ES cell lines.

Adult stem cells
Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues. Also known as somatic (from Greek Σωματικóς, of the body) stem cells, they can be found in children, as well as adults.

Adult stem cells

A great deal of adult stem cell research has focused on clarifying their capacity to divide or self-renew indefinitely and their differentiation potential.Many adult stem cells may be better classified as progenitor cells, due to their limited capacity for cellular differentiation.

Nevertheless, specific multipotent or even unipotent adult progenitors may have potential utility in regenerative medicine. The use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. In contrast with the embryonic stem cell research, more US government funding has been provided for adult stem cell research. Adult stem cells can be isolated from a tissue sample obtained from an adult. They have mainly been studied in humans and model organisms such as mice and rats.


Genetic Evidence - Transposons

Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons were also once called "jumping genes", and are examples of mobile genetic elements. Discovered by Barbara McClintock early in her career, the discovery earned her a Nobel prize in 1983. There are a variety of mobile genetic elements, and they can be grouped based on their mechanism of transposition. Class I mobile genetic elements, or retrotransposons, move in the genome by being transcribed to RNA and then back to DNA by reverse transcriptase, while class II mobile genetic elements move directly from one position to another within the genome using a transposase to "cut and paste" them within the genome. Transposons are very useful to researchers as a means to alter DNA inside of a living organism. Transposons make up a large fraction of genome sizes which is evident through the C-values of eukaryotic species.

Double Helix Lecture

Double helix (plural helices) typically consists of two congruent helices with the same axis, differing by a translation along the axis, which may or may not be half-way.
In molecular biology, the double helix refers to the structure of DNA. The structure of DNA was first published in the journal Nature by James D. Watson and Francis Crick in 1953, based upon data from Maurice Wilkins and Rosalind Franklin. Crick, Wilkins and Watson each received the Nobel Prize for their contributions to the discovery. Franklin died before her contribution could be acknowledged, and due to the fact that they cannot be awarded posthumously, never received a Nobel Prize.
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The DNA double helix is a right-handed spiral polymer of nucleic acids, held together by nucleotides which base pair together. A single turn of the helix constitutes ten nucleotides. The double helix structure of DNA contains a major groove and minor groove, the major groove being wider than the minor groove. Given the difference in widths of the major groove and minor groove, many proteins which bind to DNA do so through the wider major groove .
The order, or sequence, of the nucleotides in the double helix within a gene specifies the primary structure of a protein.

Protein Methyltransferase Inhibitors as Personalized Cancer Therapeutics

AB toxin

The AB toxins are two-component protein complexes secreted by a number of pathogenic bacteria. They can be classified as Type III toxins because they interfere with internal cell function. They are named AB toxins due to their components: the "A" component is usually the "active" portion, and the "B" component is usually the "binding" portion. The "A" subunit possesses enzyme activity, and is transferred to the host cell following a conformational change in the membrane-bound transport "B" subunit.Among the toxins produced by certain Clostridium spp. are the binary exotoxins. These proteins consist of two independent polypeptides, which correspond to the A/B subunit moieties. The enzyme component (A) enters the cell through endosomes produced by the oligomeric binding/translocation protein (B), and prevents actin polymerisation through ADP-ribosylation of monomeric G-actin
Members of the "A" binary toxin family include C. perfringens iota toxin Ia , C. botulinum C2 toxin CI, and Clostridium difficile ADP-ribosyltransferase . Other homologous proteins have been found in Clostridium spiroforme.Members of the "B" binary toxin family include the Bacillus anthracis protective antigen (PA) protein, most likely due to a common evolutionary ancestor. B. anthracis, a large Gram-positive spore-forming rod, is the causative agent of anthrax. Its two virulence factors are the poly-D-glutamate polypeptide capsule, and the actual anthrax exotoxin. The toxin comprises three factors: the protective antigen (PA); the oedema factor (EF); and the lethal factor (LF). Each is a thermolabile protein of ~80kDa. PA forms the "B" part of the exotoxin and allows passage of the "A" moiety (consisting of EF and LF) into target cells. PA protein forms the central part of the complete anthrax toxin, and translocates the B moiety into host cells after assembling as a heptamer in the membrane. The AB5 toxins are usually considered a type of AB toxin, characterized by B pentamers. Less commonly, the term "AB toxin" is used to emphasize the monomeric character of the B component.

Fighting Cancer with Magnetic Nanoparticles


Conotoxin is a neurotoxic peptides .It is isolated from the venom of marine cone snail,genus Conus,Its consist of 10 to 30 aminoacid residues,Typically have one or more dilsulfde bonds,Conotoxins have a variety of mechanisms of action like ion channels. Types and biological activities of conotoxins The number of conotoxins whose activities have been determined so far is five, and they are called the α(alpha)-, δ(delta)-, κ(kappa)-, μ(mu)-, and ω(omega)- types. Each of the five types of conotoxins attacks a different target

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* α-conotoxin inhibits acetylcholine receptors at nerves and muscles. * δ-conotoxin inhibits the inactivation of voltage-dependent sodium channels. * κ-conotoxin inhibits potassium channels. * μ-conotoxin inhibits voltage-dependent sodium channels in muscles. * ω-conotoxin inhibits N-type voltage-dependent calcium channels. Because N-type voltage-dependent calcium channels are related to algesia (sensitivity to pain) in the nervous system, ω-conotoxin has an analgesic effect: the effect of ω-conotoxin M VII A is 100 to 1000 times that of morphine.[7] Therefore a synthetic version of ω-conotoxin M VII A has found application as an analgesic drug ziconotide (Prialt).
Types of conotoxins also differ in the number and pattern of disulfide bonds [9]. The disulfide bonding network, as well as specific amino acids in inter-cysteine loops, provide the specificity of conotoxins. Omega, delta and kappa conotoxins Omega, delta and kappa families of conotoxins have a knottin or inhibitor cysteine knot scaffold. The knottin scaffold is a very special disulfide-through-disulfide knot, in which the III-VI disulfide bond crosses the macrocycle formed by two other disulfide bonds (I-IV and II-V) and the interconnecting backbone segments, where I-VI indicates the six cysteine residues starting from the N-terminus. The cysteine arrangements are the same for omega, delta and kappa families, even though omega conotoxins are calcium channel blockers, whereas delta conotoxins delay the inactivation of sodium channels, and kappa conotoxins are potassium channel blockers. Mu conotoxins Mu conotoxins have two types of cysteine arrangements, but the knottin scaffold is not observed. Mu conotoxins target the voltage-gated sodium channels[9], and are useful probes for investigating voltage-dependent sodium channels of excitable tissues

p53 Pathway Polymorphisms and Predisposition to Cancer


Tonsillitis is an infectious inflammation of the tonsils and will often, but not necessarily, cause a sore throat and fever.

There are 3 main types of tonsillitis: acute, subacute and chronic. Acute tonsillitis can either be bacterial or viral(75%) in origin. Subacute tonsillitis (which can last between 3 weeks and 3 months) is caused by the bacterium Actinomyces. Chronic tonsillitis, which can last for long periods if not treated, is almost always bacterial.
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Symptoms of tonsillitis include a severe sore throat (which may be experienced as referred pain to the ears), painful/difficult swallowing, headache, fever and chills, and loss of voice. Tonsillitis is characterized by signs of red, swollen tonsils which may have a purulent exudative coating of white patches (i.e. pus). In addition there may be enlarged and tender neck cervical lymph nodes.


Bacterial tonsillitis may be caused by Group A streptococcal bacteria, resulting in strep throat. Viral tonsillitis may be caused by numerous viruses such as the Epstein-Barr virus (the cause of infectious mononucleosis) or the Adenovirus.

Sometimes, tonsillitis is caused by a superinfection of spirochaeta and treponema, in this case called Vincent's angina or Plaut-Vincent angina.

Although tonsillitis is associated with infection, it is currently unknown if the swelling and other symtoms are caused by the infectious agents themselves, or by the host immune response to these agents. Tonsillitis may be a result of aberrant immune responses to the normal bacterial flora of the nasopharynx.

Treatments of tonsillitis consist of pain management medications and lozenges. If the tonsillitis is caused by bacteria, then antibiotics are prescribed with Penicillin being most commonly used. Erythromycin is used for patients allergic to penicillin.

In many cases of tonsillitis, the pain caused by the inflamed tonsils warrants the prescription of topical anesthetics for temporary relief. Viscous lidocaine solutions are often prescribed for this purpose.

Ibuprofen or other analgesic can help to decrease the edema and inflammation which will ease the pain and allow the patient to swallow liquids sooner.

When tonsillitis is caused by a virus, the length of illness depends on which virus is involved. Usually, a complete recovery is made within one week, however some rare infections may last for up to two weeks.

Chronic cases may indicate tonsillectomy (surgical removal of tonsils) as a choice for treatment

Additionally, gargling with a solution of warm water and salt may reduce pain and swelling

Viral Life Cycle

Viral replication is the term used by virologists to describe the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. From the perspective of the virus, the purpose of viral replication is to allow production and survival of its kind. By generating abundant copies of its genome and packaging these copies into viruses, the virus is able to continue infecting new hosts. Replication between viruses is greatly varied and depends on the type of genes involved.

Delivering a single virus to a cell allows the virus to infect the cell, replicate, and give rise to many progeny viruses. These viruses can then infect many neighboring cells.

Drug Receptors


Schizophrenia is a chronic, severe, and disabling brain disorder that has been recognized throughout recorded history. It affects about 1 percent of Americans.
People with schizophrenia may hear voices other people don't hear or they may believe that others are reading their minds, controlling their thoughts, or plotting to harm them. These experiences are terrifying and can cause fearfulness, withdrawal, or extreme agitation. People with schizophrenia may not make sense when they talk, may sit for hours without moving or talking much, or may seem perfectly fine until they talk about what they are really thinking. Because many people with schizophrenia have difficulty holding a job or caring for themselves, the burden on their families and society is significant as well.

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Available treatments can relieve many of the disorder's symptoms, but most people who have schizophrenia must cope with some residual symptoms as long as they live. Nevertheless, this is a time of hope for people with schizophrenia and their families. Many people with the disorder now lead rewarding and meaningful lives in their communities. Researchers are developing more effective medications and using new research tools to understand the causes of schizophrenia and to find ways to prevent and treat it.

This brochure presents information on the symptoms of schizophrenia, when the symptoms appear, how the disease develops, current treatments, support for patients and their loved ones, and new directions in research.

Psychotic symptoms (such as hallucinations and delusions) usually emerge in men in their late teens and early 20s and in women in their mid-20s to early 30s. They seldom occur after age 45 and only rarely before puberty, although cases of schizophrenia in children as young as 5 have been reported. In adolescents, the first signs can include a change of friends, a drop in grades, sleep problems, and irritability. Because many normal adolescents exhibit these behaviors as well, a diagnosis can be difficult to make at this stage. In young people who go on to develop the disease, this is called the "prodromal" period.

Research has shown that schizophrenia affects men and women equally and occurs at similar rates in all ethnic groups around the world.

Neurons and How They Work

Neurons are electrically excitable cells in the nervous system that process and transmit information. Neurons are the core components of the brain, and spinal cord in vertebrates and ventral nerve cord in invertebrates, and peripheral nerves.

Neurons are usually considered permanently amitotic (they do not divide) however, recent research shows that they do indeed undergo adult neurogenesis.Neurons are typically composed of a soma, or cell body, a dendritic tree and an axon. The majority of vertebrate neurons receive input on the cell body and dendritic tree, and transmit output via the axon. However, there is great heterogeneity throughout the nervous system and the animal kingdom, in the size, shape and function of neurons.

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Neurons communicate via chemical and electrical synapses, in a process known as synaptic transmission. The fundamental process that triggers synaptic transmission is the action potential, a propagating electrical signal that is generated by exploiting the electrically excitable membrane of the neuron. This is also known as a wave of depolarization.

Anatomy and histology
Neurons are highly specialized for the processing and transmission of cellular signals. Given the diversity of functions performed by neurons in different parts of the nervous system, there is, as expected, a wide variety in the shape, size, and electrochemical properties of neurons. For instance, the soma of a neuron can vary from 4 to 100 micrometers in diameter.
  • The soma is the central part of the neuron. It contains the nucleus of the cell, and therefore is where most protein synthesis occurs. The nucleus ranges from 3 to 18 micrometers in diameter.
  • The dendrites of a neuron are cellular extensions with many branches, and metaphorically this overall shape and structure is referred to as a dendritic tree. This is where the majority of input to the neuron occurs. Information outflow (i.e. from dendrites to other neurons) can also occur, but not across chemical synapses; there, the backflow of a nerve impulse is inhibited by the fact that an axon does not possess chemoreceptors and dendrites cannot secrete neurotransmitter chemicals. This unidirectionality of a chemical synapse explains why nerve impulses are conducted only in one direction.
  • The axon is a finer, cable-like projection which can extend tens, hundreds, or even tens of thousands of times the diameter of the soma in length. The axon carries nerve signals away from the soma (and also carry some types of information back to it). Many neurons have only one axon, but this axon may - and usually will - undergo extensive branching, enabling communication with many target cells. The part of the axon where it emerges from the soma is called the axon hillock. Besides being an anatomical structure, the axon hillock is also the part of the neuron that has the greatest density of voltage-dependent sodium channels. This makes it the most easily-excited part of the neuron and the spike initiation zone for the axon: in neurological terms it has the most negative hyperpolarized action potential threshold. While the axon and axon hillock are generally involved in information outflow, this region can also receive input from other neurons.
  • The axon terminal contains synapses, specialized structures where neurotransmitter chemicals are released in order to communicate with target neurons.

Although the canonical view of the neuron attributes dedicated functions to its various anatomical components, dendrites and axons often act in ways contrary to their so-called main function.

Axons and dendrites in the central nervous system are typically only about one micrometer thick, while some in the peripheral nervous system are much thicker. The soma is usually about 10–25 micrometers in diameter and often is not much larger than the cell nucleus it contains. The longest axon of a human motoneuron can be over a meter long, reaching from the base of the spine to the toes. Sensory neurons have axons that run from the toes to the dorsal columns, over 1.5 meters in adults. Giraffes have single axons several meters in length running along the entire length of their necks. Much of what is known about axonal function comes from studying the squid giant axon, an ideal experimental preparation because of its relatively immense size (0.5–1 millimeters thick, several centimeters long).

Structural classification


Most neurons can be anatomically characterized as:
  • Unipolar or pseudounipolar: dendrite and axon emerging from same process.
  • Bipolar: axon and single dendrite on opposite ends of the soma.
  • Multipolar: more than two dendrites:
  • Golgi I: neurons with long-projecting axonal processes; examples are pyramidal cells, Purkinje cells, and anterior horn cells.
  • Golgi II: neurons whose axonal process projects locally; the best example are the granule cells.


Furthermore, some unique neuronal types can be identified according to their location in the nervous system and distinct shape. Some examples are:

  • Basket cells, neurons with dilated and knotty dendrites in the cerebellum.
  • Betz cells, large motor neurons.
  • Medium spiny neurons, most neurons in the corpus striatum.
  • Purkinje cells, huge neurons in the cerebellum, a type of Golgi I multipolar neuron.
  • pyramidal cells, neurons with triangular soma, a type of Golgi I.
  • Renshaw cells, neurons with both ends linked to alpha motor neurons.
  • Granule cells, a type of as Golgi II neuron.
  • anterior horn cells, motoneurons located in the spinal cord.

Functional classification
  • Afferent neurons convey information from tissues and organs into the central nervous system and are sometimes also called sensory neurons.
  • Efferent neurons transmit signals from the central nervous system to the effector cells and are sometimes called motor neurons.
  • Interneurons connect neurons within specific regions of the central nervous system.

Afferent and efferent can also refer generally to neurons which, respectively, bring information to or send information from the brain region.

Action on other neurons
  • Excitatory neurons excite their target neurons. Excitatory neurons in the central nervous system, including the brain, are often glutamatergic. Neurons of the peripheral nervous system, such as spinal motoneurons that synapse onto muscle cells, often use acetylcholine as their excitatory neurotransmitter. However, this is just a general tendency that may not always be true. It is not the neurotransmitter that decides excitatory or inhibitory action, but rather it is the postsynaptic receptor that is responsible for the action of the neurotransmitter.
  • Inhibitory neurons inhibit their target neurons. Inhibitory neurons are often interneurons. The output of some brain structures (neostriatum, globus pallidus, cerebellum) are inhibitory. The primary inhibitory neurotransmitters are GABA and glycine.
  • Modulatory neurons evoke more complex effects termed neuromodulation. These neurons use such neurotransmitters as dopamine, acetylcholine, serotonin and others.

Discharge patterns

Neurons can be classified according to their electrophysiological characteristics:
  • Tonic or regular spiking. Some neurons are typically constantly (or tonically) active. Example: interneurons in neurostriatum.
  • Phasic or bursting. Neurons that fire in bursts are called phasic.
  • Fast spiking. Some neurons are notable for their fast firing rates, for example some types of cortical inhibitory interneurons, cells in globus pallidus.
  • Thin-spike. Action potentials of some neurons are more narrow compared to the others. For example, interneurons in prefrontal cortex are thin-spike neurons.

Neurotransmitter released

Some examples are
  • cholinergic neurons
  • GABAergic neurons
  • glutamatergic neurons
  • dopaminergic neurons
  • 5-hydroxytryptamine neurons (5-HT; serotonin)


Neurons communicate with one another via synapses, where the axon terminal of one cell impinges upon a dendrite or soma of another (or less commonly to an axon). Neurons such as Purkinje cells in the cerebellum can have over 1000 dendritic branches, making connections with tens of thousands of other cells; other neurons, such as the magnocellular neurons of the supraoptic nucleus, have only one or two dendrites, each of which receives thousands of synapses. Synapses can be excitatory or inhibitory and will either increase or decrease activity in the target neuron. Some neurons also communicate via electrical synapses, which are direct, electrically-conductive junctions between cells.

In a chemical synapse, the process of synaptic transmission is as follows: when an action potential reaches the axon terminal, it opens voltage-gated calcium channels, allowing calcium ions to enter the terminal. Calcium causes synaptic vesicles filled with neurotransmitter molecules to fuse with the membrane, releasing their contents into the synaptic cleft. The neurotransmitters diffuse across the synaptic cleft and activate receptors on the postsynaptic neuron.

The human brain has a huge number of synapses. Each of the 1011 (one hundred billion) neurons has on average 7,000 synaptic connections to other neurons. It has been estimated that the brain of a three-year-old child has about 1016 synapses (10 quadrillion). This number declines with age, stabilizing by adulthood. Estimates vary for an adult, ranging from 1015 to 5 x 1015 synapses (1 to 5 quadrillion).

Mechanisms for propagating action potentials
The cell membrane in the axon and soma contain voltage-gated ion channels which allow the neuron to generate and propagate an electrical impulse (an action potential). Substantial early knowledge of neuron electrical activity came from experiments with squid giant axons. In 1937, John Zachary Young suggested that the giant squid axon can be used to study neuronal electrical properties.As they are much larger than human neurons, but similar in nature, it was easier to study them with the technology of that time. By inserting electrodes into the giant squid axons, accurate measurements could be made of the membrane potential.

Electrical activity can be produced in neurons by a number of stimuli. Pressure, stretch, chemical transmitters, and electrical current passing across the nerve membrane as a result of a difference in voltage can all initiate nerve activity.

The narrow cross-section of axons lessens the metabolic expense of carrying action potentials, but thicker axons convey impulses more rapidly. To minimize metabolic expense while maintaining rapid conduction, many neurons have insulating sheaths of myelin around their axons. The sheaths are formed by glial cells: oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. The sheath enables action potentials to travel faster than in unmyelinated axons of the same diameter, whilst using less energy. The myelin sheath in peripheral nerves normally runs along the axon in sections about 1 mm long, punctuated by unsheathed nodes of Ranvier which contain a high density of voltage-gated ion channels. Multiple sclerosis is a neurological disorder that results from demyelination of axons in the central nervous system.

Some neurons do not generate action potentials, but instead generate a graded electrical signal, which in turn causes graded neurotransmitter release. Such nonspiking neurons tend to be sensory neurons or interneurons, because they cannot carry signals long distances.

Neurons in the brain
The number of neurons in the brain varies dramatically from species to species. One estimate puts the human brain at about 100 billion (1011) neurons and 100 trillion (1014) synapses. By contrast, the nematode worm Caenorhabditis elegans has just 302 neurons making it an ideal experimental subject as scientists have been able to map all of the organism's neurons. By contrast, the fruit fly Drosophila melanogaster has around 300,000 neurons (which do spike) and exhibits many complex behaviors. Many properties of neurons, from the type of neurotransmitters used to ion channel composition, are maintained across species, allowing scientists to study processes occurring in more complex organisms in much simpler experimental systems.

Neurologic Diseases

Alzheimer's disease: Alzheimer's disease (AD), also known simply as Alzheimer's, is a neurodegenerative disease characterized by progressive cognitive deterioration together with declining activities of daily living and neuropsychiatric symptoms or behavioral changes. The most striking early symptom is loss of short-term memory (amnesia), which usually manifests as minor forgetfulness that becomes steadily more pronounced with illness progression, with relative preservation of older memories. As the disorder progresses, cognitive (intellectual) impairment extends to the domains of language (aphasia), skilled movements (apraxia), recognition (agnosia), and functions such as decision-making and planning get impaired.

Parkinson's disease: Parkinson's disease (also known as Parkinson disease or PD) is a degenerative disorder of the central nervous system that often impairs the sufferer's motor skills and speech. Parkinson's disease belongs to a group of conditions called movement disorders. It is characterized by muscle rigidity, tremor, a slowing of physical movement (bradykinesia), and in extreme cases, a loss of physical movement (akinesia). The primary symptoms are the results of decreased stimulation of the motor cortex by the basal ganglia, normally caused by the insufficient formation and action of dopamine, which is produced in the dopaminergic neurons of the brain. Secondary symptoms may include high level cognitive dysfunction and subtle language problems. PD is both chronic and progressive.

Myasthenia Gravis: Myasthenia gravis is a neuromuscular disease leading to fluctuating muscle weakness and fatigability. Weakness is typically caused by circulating antibodies that block acetylcholine receptors at the post-synaptic neuromuscular junction, inhibiting the stimulative effect of the neurotransmitter acetylcholine. Myasthenia is treated with immunosuppressants, cholinesterase inhibitors and, in selected cases, thymectomy.

Demyelination is the act of demyelinating, or the loss of the myelin sheath insulating the nerves. When myelin degrades, conduction of signals along the nerve can be impaired or lost, and the nerve eventually withers. This leads to certain neurodegenerative disorders like multiple sclerosis, chronic inflammatory demyelinating polyneuropathy.

Axonal Degeneration
Although most injury responses include a calcium influx signaling to promote resealing of severed parts, axonal injuries initially lead to acute axonal degeneration (AAD), which is rapid separation of the proximal and distal ends within 30 minutes of injury. Degeneration follows with swelling of the axolemma, and eventually leads to bead like formation. Granular disintegration of the axonal cytoskeleton and inner organelles occurs after axolemma degradation. Early changes include accumulation of mitochondria in the paranodal regions at the site of injury. Endoplasmic reticulum degrades and mitochondria swell up and eventually disintegrate. The disintegration is dependent on Ubiquitin and Calpain proteases (caused by influx of calcium ion), suggesting that axonal degeneration is an active process. Thus the axon undergoes complete fragmentation. The process takes about roughly 24 hrs in the PNS, and longer in the CNS. The signaling pathways leading to axolemma degeneration are currently unknown.

From axons to tracts: A journey through the brain's wiring

Increased Receptor Sensitivity Video

In LTP, it is now known that the postsynaptic neuron becomes more sensitive to neurotransmitter in a variety of ways. One way is that phophorylation of the glutamate receptor causes it to pass more excitatory ions upon subsequent stimulation

Protemist DT - Automated Wheat Germ Protein Synthesizer

GST-fusion Protein Purification

1. In a 125 mL flask containing 10 mL of LB and 10 mL ofAmpicillin (100 mg/mL -for pGEX vectors, Life Technologies), transfer a single colony and Grow overnight
2. Transfer 5 mL of ON culture to a 250 mL flask with 45 mL of LB and 50 mL of Ampicillin (100 mg/mL)

3. Incubate at 28-37oC until A600 reaches 0.7 (best) - 0.8 (up to ~1.0). Induce with 10 mL of 1M IPTG/50 mL culture*. Incubate at 28oC (can also try at 37oC) for >2 hours at 250 rpms (longer incubations are OK if the GST-protein is stable and/or growing in BL21-DE3 proteolysis- cells)

*NOTE: varying the concentrations of IPTG may increase the yield of your protein (5 - 25mL of 1M IPTG/50 mL culture)
NOTE: growth of the expressed protein at 28oC after induction is supposed to reduce aggregation of the fusion protein
4. Centrifuge at 8,000 g for 10 mins at 4oC and discard the supernatant
Note: the pellet can be stored at -80oC for weeks
5. Resuspend pellet with 10 mL of cold buffer + 10 µL of 100 mM PMSF at 4oC.

BEST: in addition to the PMSF, other protease inhibitors should be added. A good cocktail of protease inhibitors is Complete (from Roche). This cocktail comes as a tablet that can be dissolved in 10 mL of buffer and provides good inhibition against 5 of the main proteases (at pH 7.8)
6. Lyze cells by 2-3 passages through a French press (4oC) at a pressure no lower than 1100 psi (keep pressure around 1100-1200 rpms) (cell lysis can be detected by clearing of suspension unless a high concentration of inclusion bodies is present)
NOTE: other means of cell lysis, like sonication, are not as good as a French press. In my hands, purifying the proteins I have purified, French-pressed samples yield more purified protein when compared to other extraction procedures. In a study I performed, 10 times more protein was obtained using a French-pressed sample as compared to a sonicated one

7. Add 0.5 mL of 20% Triton X-100/10 mL of lysate (to 1%Triton X-100). Mix on a rocker for 30-60 mins at 4oC (longer incubations may reduce the amount of inclusions bodies)
8. Centrifuge at 12,000 g for 20 minutes at 4oC and save the supernatant (can be stored at -80oC). This is termed total soluble protein = T

Optional: Inclusion Body (IB) proteins can be solubilized. Add 1 mL/50 mL culture pellet of IB buffer, sonicate (2-4x for 10-15 secs in a water bath somicator), resuspend and mix on a shaker for >30-60 mins at 4oC. Centrifuge at 12,000 g for 15 mins and save IB supernatant
9. Equilibrate a 50% slurry of Glutathione Sepharose 4B (Pharmacia Biotech, catalog # 27-4574-01) with PBS for 10 mins, spin for 2 mins at 500 g and discard wash. Add 10 mL of T / 2 mL of 50% slurry
Glutathione Sepharose 4B: store in 20% ethanol. 200-400 mmol Glutathione Sepharose per g dried substance. Spherical beads, 45-165 mm wet diameter. Spacer arm: 12 atoms (10 carbons)
10. Rock for >20 minutes at RT

11. Centrifuge at 500 g for 1 min. Save an aliquot of solution (Flow Through = FT)
12. Aspirate off supernatant withough getting too close to the beads and wash 4 times with 1X PBS/Wash solution each for <1 minute, namely invert the tube until the beads are resuspended (centrifuge for 2 mins at 500 g to pellet beads).

Cadherins Animation

Cadherins mediate cell adhesion and play a fundamental role in normal development. They participate in the maintenance of proper cell-cell contacts. Cadherins typically consist of five tandem repeated extracellular domains, a single membrane-spaning segment and a cytoplasmic region. Cadherins depend on calcium for their function: removal of calcium abolishes adhesive activity and renders cadherins vunerable to proteases.


Cadherins generally mediate monotypic cell-cell adhesion although heterotypic binding between different cadherin molecules is possible. They act as both receptor and ligand. They are responsible for the selective cell-cell adhesion or cell sorting which is necessary to allocate different cell types to their proper positions during development. They also play a fundamental role in maintaining the integrity of multicellular structures. During embryonic morphogenesis, the expression of multiple members of the cadherin family is spatio-temporally regulated, and correlates with a variety of morphogenetic events that involve cell aggregation or disaggregation.

The role of cadherins in these processes has been demonstrated by the use of anti-cadherin monoclonal antibodies, which, when added to embyonic tissues, cause a severe distortion of structure which results in dissociation of the tissue into small clusters or even single cells.


The cadherins are synthesized as a precursor polypeptide which requires a series of post-translational modifications (glycosylation, phosphorylation and proteolytic cleavage) to form a protein which is between 723 and 748 amino acids long. The extracelluar domain contains 3-5 internal repeats of approximately 110 amino acids. Repeats 1-3 contain the putative Ca2+ binding site motif DXNDN or DXD, resembling the Ca2+ binding lops of the so-called EF-hand domains. Another high conserved cluster with the motif LDREXXXXYXL is also found with the first three repeats.

The N-terminal 113 amino acids which contain a conserved HAV sequence have been shown to be important in ligand binding and specificity. The extracellular domain is anchored to the cell membrane by a transmembrance domain of approximately 24 amino acids. The short cytoplasmic domain is the most highly conserved region of homology between cadherins and is particularly important for cadherin function
Cadherins are differentially expressed during development and in adult organs. Since many cell types express multiple cadherin subclasses simultaneously (the combination differs with cell type), it can be inferred that the adhesion properities of individual cells are thus governed by varying the combinations of cadherins. There is also recent evidence to suggest that altered expression of cadherins may be involved in invasion and metastasis of tumour cells.
On the cell surface, cadherins tend to be concentrated at cell-cell junctions. Here they are structurally associated with cortical actin bundles. The cytoplasmic domain has been shown to be associated with cytoplasmic proteins termed catenins. Deletion of the cytoplasmic domain destroys these interactions and also eliminates cadherin function. Recently src, yes, and lyn gene products of the src proto-oncogene family have been found expressed at cell-cell junctions. These kinases may be responsible for cadherin phosphorylation and this raises the possibly that cadherin mediated cell junctions might be used for intercellular signalling

Signal Recognition Particle (SRP)

Signal recognition particle (SRP) is a ribonucleoprotein (protein-RNA complex) that recognizes and transports specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes.

The SRP is found in the cytosol. In eukaryotes, it binds to the endoplasmic reticulum signal sequence found in an emerging secretory protein and subsequently delivers the protein along with the ribosome to the membrane of the endoplasmic reticulum. When SRP binds the endoplasmic reticulum signal sequence, it also binds the ribosome and thereby pauses translation until the entire SRP-bound translation complex is transported to the endoplasmic reticulum, where translation resumes.
In the GTP-bound state, it recognizes an endoplasmic reticulum signal sequence of eight or more nonpolar amino acid residues at its center. It transiently binds to the endoplasmic reticulum signal sequence in a nascent protein, to the large ribosomal unit, and to the SRP receptor.
SRP and its receptor initiate the transfer of the nascent chain across the endoplasmic reticulum membrane. Once the chain reaches the translocon, SRP and its receptor both hydrolyze their bound GTP to GDP, then dislocate allowing translation to restart.

Histone Modification Animation

Histones are subject to a wide variety of posttranslational modifications including but not limited to, lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation (Vasquero 2003). These modifications occur primarily within the histone amino-terminal tails protruding from the surface of the nucleosome as well as on the globular core region (Cosgrove 2004).

Histone modifications are proposed to affect chromosome function through at least two distinct mechanisms. The first mechanism suggests modifications may alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA. The second mechanism proposes that these modifications are binding sites for protein recognition modules, such as the bromodomains or chromodomains, that recognize acetylated lysines or methylated lysine, respectively.
The existence of these modifications and recognition modules led to a well established “histone code” hypothesis proposed by Strahl and Allis (2000). Overall, posttranslational modifications of histones create an epigenetic mechanism for the regulation of a variety of normal and disease-related processes.