Apoptosis Programmed Cell Death

Coined in the 1960's,apoptosis is derived from the Greek word apopiptein which means to fall off from. Apoptosis can be induced by a number of stimuli including UV damage, irradiation, drug treatment, or tumor necrosis factor. Once induced, apoptosis can, in turn, act through a number of different cell death signaling pathways.

The number of apoptosis and 'programmed cell death' related kits and reagents available to the market have increased significantly over the last few years. This is in large part the result of increasing evidence implicating the role of apoptosis in a number of significantly relevant disease processes including certain autoimmune diseases, transplantation rejection, and neurodegenerative diseases. IMGENEX offers over 200 Apoptosis related antibodies, as well as ELISA kits, caspase inhibitors, active caspase detection kits, apoptosis detection kits, and mitochondrial permeability detection kits.

Regulatory T Cells (Treg)

Early development and differentiation of nascent T cells that migrate from bone marrow to become mature, naïve T cells, which are capable of responding to antigen takes place inside the thymus. Around 10¹⁰ TCR (T cell receptor) variations are generated in developing T lymphocyte clones through a random process of somatic cell gene reorganization. During this process, often T-cells recognizing self-antigens are generated. Due to the ability of these self-reactive T-cells to elicit an autoimmune attack, they are permanently removed by the thymus through negative selection and clonal deletion. But, some of them manage to escape the thymic defenses and harbor themselves in the peripheral lymphoid organs. In periphery, T lymphocytes undergo further differentiation into effectors of various immune functions.

One of many immunotolerance mechanisms that immune system has developed to distinguish between self and non-self antigens is regulatory T cells or Tregs. These cells are recently characterized specialized T-cell subsets that actively suppress a variety of immune responses. Researchers have broadly classified Tregs into natural and adaptive Tregs. Natural Tregs are CD4⁺CD25⁺ T-cells that originate in the thymus and play a significant role in immune homeostasis and protection against autoimmunity. Adaptive Tregs are non-regulatory CD4⁺ T-cells that have up-regulated CD25 expression during pathological and inflammatory conditions such as cancers and infections.

Although the principal immunosuppressive mechanism of Tregs remains elusive, several in vivo experimental models have indicated that Tregs secrete large amounts of immunosuppressants including IL-9, IL-10 and TGF-β upon activation. These lymphokines are capable of inhibiting activation of Th1, Th2 cells and CTLs required for cell-mediated immunity, inflammation and antibody production. Certain recent experimental data and results even indicate that IL-2-IL-2R signaling is vital for development, maintenance, survival, expansion and suppressive activity of Tregs. Increased expression of certain other characteristic markers including CTLA-4, glucocorticoid-inducible tumor necrosis factor receptor (GITR) and OX40 has been identified on Tregs whose function inside these cells is still not clear. The TCRs displayed on Tregs are capable of recognizing and interacting with any peptide-MHC class II ligand having certain range of avidity. But, the contribution of TCR signaling and role of TCR-ligand interactions towards regulatory T-cell development needs to be determined.

Several elegant experiment using transgenic mice and retrovirus mediate over expression studies, researchers have identified FoxP3, a transcription factor, to be a specific molecular marker essential for the development and function of Tregs. The primary evidence regarding the involvement of FoxP3 in the development of Tregs was provided by the experiments of Sakaguchi et al, (ref ?) in patients suffering from IPEX, a rare and fatal human autoimmune disorder. In these patients, mutated FoxP3 gene causes improper development of Tregs resulting in hyperactivation of T-cells reactive to self-antigens. Recently, experiments have clearly shown that retroviral mediated introduction of FoxP3 into conventional CD4+ T-cells converts them into regulatory T-cells.

The emergence of regulatory T-cells and role of FoxP3 as a critical player in the negative control of a of various normal and pathological immune responses holds great promise for the development of novel therapies useful for the treatment of autoimmune diseases in humans. However, there are several questions that remain to be answered including the basic biology of the Tregs, various ligands responsible for thymic selection of these cells, the exact function of FoxP3 in relation with various markers present on Tregs and most importantly, the mechanisms by which Tregs exert their suppressive effects. A better understanding of manipulating FoxP3 and Tregs would enable us to harness the tremendous therapeutic potential in various clinical situations including Type I diabetes, Multiple sclerosis, GVHD, rheumatoid arthritis, allergy, and cancers.

Actin Antibody Available in Imgenex now

Actin is a ubiquitous protein involved in the formation of filaments that are major components of the cytoskeleton. It is the monomeric subunit of microfilaments, one of the three major components of the cytoskeleton, and of thin filaments which are part of the contractile apparatus in muscle cells. It is the most abundant protein in the typical eukaryotic cell, accounting for about 15% in some cell types. The protein is highly conserved, and forms a huge variety of structure in cells in concert with a huge numbers of actin binding proteins. The actin filaments interact with myosin to produce a sliding effect, which is the basis of muscular contraction and many aspects of cell motility, including cytokinesis. The individual subunits of actin are known as globular actin (G-actin) that assembles into long filamentous polymers called F-actin. Two parallel F-actin strands twist around each other in a helical formation, giving rise to microfilaments of the cytoskeleton. Microfilaments measure approximately 7 nm in diameter with a loop of the helix repeating every 37nm. Each actin protomer binds one molecule of ATP and has one high affinity site for either calcium or magnesium ions, as well as several low affinity sites. It exists as a monomer in low salt concentrations, but filaments form rapidly as salt concentration rises, with the consequent hydrolysis of ATP. Actin from many sources forms a tight complex with deoxyribonuclease (DNase I) although the significance of this is still unknown. The formation of this complex results in the inhibition of DNase I activity, and actin loses its ability to polymerise. It has been shown that an ATPase domain of actin shares similarity with ATPase domains of hexokinase and hsp70 proteins. In vertebrates there are three groups of actin isoforms: alpha, beta and gamma. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta and gamma actins co-exist in most cell types as components of the cytoskeleton and as mediators of internal cell motility. MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Further it has been suggested that members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion.

Reference:

1. Actin isoforms. Curr Opin Cell Biol. 1993 Feb;5(1):48-55 Herman IM
2. The assembly of MreB, a prokaryotic homolog of actin. The assembly of MreB, a prokaryotic homolog of actin. J Biol Chem. 2005 Jan 28;280(4):2628-35. Epub 2004 Nov 16
3. Rho GTPases and the Actin Cytoskeleton Science 23 January 1998:
   Vol. 279. no. 5350, pp. 509 – 514 Alan Hall

Akt Family: Antibodies from Imgenex

Akt family of serine/threonine-directed kinases regulates a diverse array of biological processes, including cellular survival, proliferation, glucose homeostasis, and vascular tone and are important molecules in mammalian cellular signaling. The three widely expressed isoforms of PKB (PKB, PKBß and PKB; also known as Akt1, Akt2 and Akt3, respectively) are each composed of an N-terminal PtdIns(3,4,5)P₃- and PtdIns(3,4)P₂-binding PH domain and a C-terminal kinase catalytic domain. Stimulation by numerous growth factors, cytokines, hormones and neurotransmitters can activate PKB/Akt in a phosphoinositide 3-kinase-dependent manner. Through receptor tyrosine kinases, these stimuli cause phosphoinositide 3-kinase activation, and generation of the membrane phospholipid PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 then recruits PKB/Akt to the membrane, where it becomes phosphorylated (for PKBa/Akt1) by upstream kinases, phosphoinositide-dependent kinase 1. Following the activation of PI 3-kinase, PKB isoforms are recruited from the cytosol to the plasma membrane through their interaction with PtdIns(3,4,5)P₃ and/or PtdIns(3,4)P₂ where they are thought to undergo a conformational change and become activated by phosphorylation of two residues. PKB can promote cell survival by inhibiting proteins that mediate apoptosis. Phosphorylation of BAD by PKB (and other AGC kinases) enables it to interact with 14-3-3 proteins, which prevents it from binding to Bcl-X_L and thereby suppresses apoptosis. It directly phosphorylate and inhibit the caspase proteasesm, key executioners of apoptosis. PKBbeta, an essential gene for the maintenance of normal glucose homeostasis and is likely to represent a critical intermediate in the insulin signal transduction pathway. PKB activation might inhibit apoptosis by promoting the increased expression of survival molecules or the degradation of pro-apoptotic molecules. PKB also phosphorylates and activates endothelial nitric oxide synthase, thereby promoting angiogenesis (formation of new blood vessels). Inhibition of GSK3 following its phosphorylation by PKB has also been suggested to play a role in inhibiting apoptosis in neuronal cells. Thus it plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis, which suggests it, likely to be a hot drug target for the treatment of cancer, diabetes and stroke.

ATM (Ataxia Telangiectasia Mutated) Antibodies from Imgenex

ATM, the gene product mutated in the cancer susceptibility syndrome ataxia–telangiectasia, is related to proteins involved in DNA repair and cell-cycle control. It encodes a nuclear 350 kDa phosphoprotein containing a carboxy terminus phosphatidylinositol 3-kinase (Pl-3 kinase) catalytic domain shared by members of a superfamily of large eukaryotic proteins involved in intracellular signaling, DNA-damage induced cell cycle checkpoints, DNA repair and recombination. It was discovered as mutated proteins in patients with ataxia-telagiectasia (A-T), a severe genetic disorder characterized by cerebellar degeneration, neuromotor dysfunction, chromosomal instability, immune system defects, cancer predisposition, and acute sensitivity to ionizing radiations. In undamaged cells it is present as a dimer or oligomer molecule in which the kinase domain is silent because associated with the FAT region of another ATM monomer. Following DSB formation, it rapidly autophosphorylates on residue Serine 1981, and the inactive ATM dimers are converted (dissociated) into active ATM monomers. Active phosphorylated ATM molecules interact and phosphorylate downstream proteins that affect one or more of the cell cycle checkpoints. Some of the known substrates are the p53 protein and its ubiquitin ligase, MDM2; the Nbs1 protein; the Brca1 protein, which interacts with other repair proteins; the checkpoint kinase 2, Chk2; the Rad17 protein and the chromatin remodeling protein SMC1. Phylogenetic analyses reveal that the ATM protein is most closely related to several very large proteins that define a subgroup of the PI 3-kinase family which include the Schizosaccharomyces pombe Rad3 protein and its probable Saccharomyces cerevisiae homologue, Mec1p/Esr1p. Other proteins in the ATM family are S. cerevisiae Tor1p and Tor2p and their mammalian counterpart FRAP, which function, at least in part, by controlling progression through the G1 phase of the cell cycle. The ATM gene provides instructions for making a protein that is located primarily in the nucleus of cells, where it helps control the rate at which cells grow and divide and also assists cells in recognizing damaged or broken strands of DNA. It has been suggested that it acts as a lipid kinase, and feeds the phosphorylated lipids into signaling pathways to regulate cell-cycle progression or the activity of DNA-repair components. It regulates NF-κB activity and control the transcription of many genes that play important roles in the development and function of the immune system. In the DNA-damage response pathway, it acts upstream of p53 to induce cell cycle arrest at the G1/S and G2/M boundaries and a slowing of the S-phase. Signalling by ATM involves interactions with and phosphorylation of critical molecules, including the mitotic checkpoints Chk1 and Chk2. Apart from its role in ataxia telangiectasia (AT), ATM gene mutations have also been found in T-cell prolymphocytic leukaemia patients with no family history of AT and in non-Hodgkin’s lymphomas.

New FOXP3Δ2 (Exon 2 Deleted) Specific Antibody

FOXP3 is a master regulator of immune homeostasis expressed specifically in CD4⁺ CD25⁺ T regulatory cells controlling their growth, development and function. FOXP3 significance in the normal development of Tregs is better elucidated with the fact that mutated FOXP3 results in a rare and fatal early onset autoimmune disorder in humans called XLAAD/IPEX (human immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), a condition phenotypically similar to Scurfy in mice.

FOXP3 is primarily an oligomeric, transcriptional repressor protein that belongs to the P subfamily of forkhead (FKH)-winged helix family of transcriptional factors. Members of this subfamily are forkhead (FKH) box proteins characterized by the presence of a highly conserved C-terminal winged-helix/FKH DNA binding domain (DBD) and centrally-located C2H2 zinc finger domain and leucine zipper domain. Apart from these, an additional N-terminal proline rich region is present in FOXP3, whose function is yet to be understood. Studies have shown that FOXP3 is a nuclear-localized protein that specifically trans-represses NF-AT-induced expression of cytokines and other transcriptional factors in Tregs including IL-2, IL-4, IFN-gamma and NF-κB.

However, FOXP3 is not the sole master switch regulating the origin and development of CD4⁺ CD25⁺ Tregs. Studies have confirmed the existence of splice variant forms FOXP3 that are specifically expressed in humans but are lacking in mouse. Cloning and RT-PCR analysis from mRNA of CD4⁺ CD25⁺ T regulatory cells (Allan et. al, Smith et. al) has shown that these cells express two different alternatively spliced variant forms of FOXP3. While the FOXP3Δ2 variant had a deleted 105bp exon2 region, there was another FOXP3Δ2, Δ7 variant that had an additional 81bp exon7 deletion apart from exon2 deletion. The predicted molecular weight of this FOXP3Δ2 is ~4KDa lower than the molecular weight of FOXP3. Transient transfection assays using Jurkat cells suggest that the FOXP3Δ2 is novel splice variant that functions as a transcriptional repressor protein and acts in cohort with FOXP3 causing a significant suppression of cytokines and up-regulating the expression of various Treg-associated markers.

The existence of the splice variant forms of FOXP3 protein suggests an additional level of complexity related to the biology of FOXP3. A lot research needs to be done so as to elucidate the physiological and functional importance of FOXP3 splice variant forms towards maintaining immune homeostasis in Tregs and preventing autoimmune disorders.

Continuing its efforts towards supporting research on FOXP3, IMGENEX has come up with an antibody that specifically recognizes the FOXP3Δ2 splice variant.

Improved RNA interference kits launched by Imgenex

RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has been observed in all eukaryotes, from yeast to mammals. The RNAi pathway is thought to be an ancient mechanism for protecting the host and its genome against viruses and rogue genetic elements that use double-stranded RNA (dsRNA) in their life cycles. They have also been shown to play a role not only in mRNA and dsRNA stability/degradation, but also in regulation of translation, transcription, chromatin structure, and genome integrity. In plants and animals, RNA silencing has been adapted to play a critical role in regulation of cell growth and differentiation using a class of small RNAs. In the RNA interference process, the dsRNAs get processed into 20-25 nucleotide (nt) small RNAs by an RNase III-like enzyme called Dicer. Then, the small RNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The small RNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. The small RNAs that provide target specificity to the silencing machinery includes short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and microRNAs (miRNAs) and is distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two distinct strands of perfectly base-paired RNA, while miRNAs originate from a single, long transcript that forms imperfectly base-paired hairpin structures. siRNAs were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA; however, endogenous sources of siRNAs have now been recognized. The endogenous siRNAs are derived from repetitive sequences within the genome, and are termed repeat-associated siRNAs, or rasiRNAs. miRNAs were discovered through their critical roles in development and cellular regulation, and represent a large class of evolutionarily conserved RNAs. miRNAs have always been recognized as being of endogenous origin. RNA interference has emerged as a natural mechanism for silencing gene expression over the past decade. This ancient cellular antiviral response can be harnessed to allow specific inhibition of the function of any chosen target genes, including those involved in causing diseases such as cancer, AIDS, and hepatitis. It is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Last but not the least the technology can be harnessed as a novel therapeutic agent and is suitable for combating viral diseases, cancers and inflammatory diseases.

Imgenex (San Diego) recently launched the pSuppressorAdeno construction kit for adenovirus mediated gene knockdown. The kit provides the ability to infect a broad range of cell types, including many primary cell lines as well as dividing and nondividing cells, according to a company official. The kit also offers the flexibility to validate sequences using the nonviral expression plasmid prior to construction of adenoviruses, notes Sujay K. Singh, Ph.D., president and CEO of Imgenex, which markets plasmid-based RNA interference (RNAi) products. “One of the greatest advantages is the ability of recombinant adenovirus vectors to reduce gene expression both in vitro and in vivo,” he adds. RNAi, initially considered a bizarre attribute of petunias and later a gene-silencing mechanism in worms, is creating a stir as one of the hottest new technologies in molecular biology. It is revolutionizing the field of functional genomics.