<p> Simian Virus 40 (SV40) is a well-characterized virus whose small circular DNA genome is organized into chromatin and, as a consequence, undergoes many of the same biological processes observed in cellular chromatin. SV40 early transcription is repressed when the product of early transcription, T-antigen, binds to its cognate regulatory sequence, Site I, in the promoter of the SV40 minichromosome. We have subsequently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication.</p><p> In order to determine the capacity of SV40 epigenetic regulation, we have analyzed SV40 chromatin from minichromosomes and virions for the presence of modified histones using various ChIP techniques and correlated these modifications with specific biological effects on the SV40 life cycle. Since repression is frequently epigenetically marked by the introduction of specific forms of methylated histone H3, we characterized the methylation of H3 tails during transcription and replication in wild-type SV40 minichromosomes and mutant minichromosomes which did not repress T-antigen expression. While repressed minichromosomes following replication were clearly marked with H3K9me1 and H3K4me1, minichromosomes repressed during early transcription were not similarly marked. Instead repression of early transcription was marked by a significant reduction in the level of H3K9me2. The replication dependent introduction of H3K9me1 and H3K4me1 into wild-type SV40 minichromosomes was also observed when replication was inhibited with aphidicolin.</p><p> We observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.</p><p> Our results demonstrate that, like its cellular counterpart, SV40 chromatin is capable of passing biologically relevant transgenerational epigenetic information between infections. Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a 'proof reading' mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.</p>

<p> Apolipoprotein (apo) E3 (299 residues, &sim;34 kDa) and apoAI (243 residues, &sim;28 kDa) are exchangeable apolipoproteins that play a dominant role in regulating plasma cholesterol levels and are considered anti-atherogenic. The N-terminal (NT) domain of apoE3 mediates cellular uptake and clearance of plasma lipoproteins through the low density lipoprotein lipoprotein receptor (LDLr) family. The C-terminal (CT) domain of apoE3 lowers cholesterol levels through its ability to promote cholesterol efflux via ABCA1 (ATP binding cassette transporter AI), thereby mediating reverse cholesterol transport from peripheral tissues such as macrophages back to the liver. ApoAI is able to interact with phospholipid vesicles with high affinity and exhibits anti-atherogenic properties through its participation in the reverse cholesterol transport (RCT) pathway with high-density lipoprotein (HDL) and lecithin cholesterol acyltransferase (LCAT). The objective of this study was to determine the conformation and function of domain swapped chimeras of apoE3 and apoAI. Two domain swapped chimeras were generated: apoE3/apoAI and apoAI/apoE. The &alpha;-helical content of the chimeras were comparable to that of the parent proteins. Chemical denaturation studies of the chimeras revealed an unfolding profile that primarily follows the NT-domain of parents. While the apoAI/apoE chimera possessed lipid binding ability similar to its apoAI parent, the apoE3/apoAI chimera showed significant increase in lipid binding ability compared to apoE3. Whereas apoE3/apoAI elicits the ability to bind to the LDLr, apoAI/apoE did not. Lastly, both chimeras promoted ABCA1 mediated cholesterol efflux from J774 macrophages. These results show that CT of apoAI can promote lipid binding of apoE3, while CT of apoE3 can improve cholesterol efflux ability of apoAI. These findings contribute significantly to the development of therapeutic chimeras focused on reducing blood cholesterol levels.</p>

<p> Nitrogen (N) is a chemical constituent for almost all biological molecules including proteins, DNA, RNA, lipids and is therefore vital for life. The ultimate source of nitrogen is the atmospheric dinitrogen (N₂) but that only becomes bioavailable through a process of nitrogen fixation, the process that converts N₂ to ammonia (NH₃). The industrial Haber-Bosch process and biological nitrogen fixation account for the majority of nitrogen fixed every year. However, due to its high temperature, pressure and fossil fuel requirements, Haber-Bosch is an expensive process. Every year, approximately 3% of the global energy demand is used to manufacture ammonia through Haber-Bosch process. On the other hand, biological systems produce ammonia by reducing dinitrogen at ambient temperature and pressure using an anaerobic enzyme called nitrogenase. Research in understanding the mechanism of nitrogenase could eventually allow researchers to mimic the enzyme and fix nitrogen efficiently at standard temperature and pressure. </p><p> In this research, nitrogenase of <i>Azotobacter vinelandii</i> was studied to understand the mechanism of delivery of electrons/protons to the active site and how these accumulated reducing equivalents are used for substrates reduction. Through a series of studies, it has been demonstrated that the electrons and protons are added to the active site in a concerted manner which are then stored as bridging hydrides. The accumulated hydrides are used in four different mechanisms, namely reductive elimination, hydride protonolysis, migratory insertion and proton coupled electron transfer, to catalyze the reduction of varieties of unsaturated molecules. This fundamental understanding of molecular detail of nitrogenase catalysis could eventually help in development of more efficient, robust and selective catalysts.</p>

No description available.

When cultured in vitro, human embryonic stem cells (hESCs) acquire genetic abnormalities that have slowed their therapeutic use. As hESCs have a “leaky” G1/S boundary, the pressure of ensuring genetic integrity falls on the G2/M checkpoint, which can be activated by failed chromosomal decatenation (among other stimuli). It is hypothesized that hESCs have a deficient decatenation checkpoint, but little data supports this. Evidence suggests that the ataxia telangiectasia mutated (ATM) kinase controls the G2/M decatenation and DNA damage checkpoints, though previous reports are conflicting on this point. My work demonstrates that inhibition of decatenation activates ATM and arrests hESCs in G2. Pharmacologic inhibition of ATM (ATMi) abrogates this arrest, allowing hESCs to enter mitosis. Live cell imaging studies reveal that ATMi increases the time it takes to complete mitosis. Culture of cells under ATMi causes a gain of DNA content, which is reversed once ATMi is relieved. BRCA1, a known target of ATM, is also involved in the G2/M checkpoint. Experimental evidence reveals that activated ATM phosphorylates BRCA1, preventing Aurora A from interacting with and phosphorylating BRCA1 on S308, a modification necessary for mitotic entry. Together, this data illuminates a novel pathway by which ATM activation mediates G2 arrest in hESCs.

The intermediate filament gene family, composed of six classes, shows both tissue and development-specific expression. Vimentin is a unique member of the intermediate filament protein family: although it is expressed in cells of mesenchymal origin, vimentin may also be expressed with other intermediate filament proteins during early stages of development. In some cases, as differentiation continues, vimentin is normally down-regulated whereas other intermediate filament proteins, like desmin, glial fibrillary acidic protein, or neurofilaments are turned on in muscle, glial cells, or neurons, respectively. In some metastatic cancers, including breast and prostate cancers, vimentin is aberrantly expressed, despite the embryonic origin of the metastatic cell. From a Northern analysis (Thompson et al., 1992; Stover et al., 1994), it was determined that vimentin mRNA is highly abundant in the metastatic breast carcinoma cell line, MDA-MB-231, while in the nonmetastatic breast carcinoma cell line, MCF-7, no vimentin mRNA is present. A central question to metastasis is, how is the vimentin gene expressed in the metastatic but not the nonmetastatic cancer cell? Several cis-acting elements localized to the 5'-end of the chicken vimentin gene and trans-acting factors important in the regulation of vimentin gene expression have been identified. By sequence homology, comparable elements can be found in the human vimentin gene. Most notable of these is a unique silencer element and an overriding element, referred to as an antisilencer. These elements bind a 90 kDa and 120 kDa protein, respectively, in chicken, mouse, and human nuclear extracts. Previously, the silencer factor was found to be missing in nuclear extracts from a metastatic breast cancer cell line MDA-MB-231 but abundant in the nonmetastatic counterpart MCF-7 (Stover et al., 1994). The antisilencer exhibits the opposite pattern. Here, various combinations of these elements have been fused to the bacterial chloramphenicol acetyltransferase reporter gene, CAT. Transcriptional activity was then compared in the different breast cancer cell lines in order to try and understand how vimentin is expressed in the metastatic but not the nonmetastatic cancer cell.

The hepatitis c virus (HCV) core gene has been chemically synthesized and used to direct the expression of core protein in Escherichia coli (E.coli). When cloned downstream of the inducible T7 promotor the core gene directed the expression of a soluble protein with a molecular weight of 22 kDa. Under native conditions the protein behaved in a manner consistent with the formation of a multimeric structure, which may represent assembled core particles. Further, when examined by electron microscopy a heterogeneous mixture of nucleocapsid-like particles were visible. Core protein was specifically recognized by antibodies present in HCV infected serum, suggesting that this protein may be useful as a diagnostic tool for detecting HCV infection. To circumvent the difficulty in purifying this protein in significant quantities a core-polyhistidine fusion gene was constructed. This protein was chromatography, readily purified, using nickel under denaturing conditions. chelation. Studies are currently on-going to ascertain the conditions required for refolding this protein, and the suitability of this protein to serve as a diagnostic tool. In a second study, the major core protein (p26) of the lentivirus equine infectious anaemia (EIAV) was expressed in E.coli and purified to >95% homogeneity. Circular dichroism spectroscopy revealed that p26 exhibits the following assignment of secondary structural elements; 40% alpha helix, 22% beta sheet, 10% beta turn, and 28% random coil. It has been determined that p26 contains a single free cysteine residue (Cys48), and an intramolecular disulfide bond between cysteine residues 198 and 213. Data acquired by circular dichroism spectroscopy and fluorescence spectroscopy indicate that this disulfide bond plays a critical role in maintaining the structure of p26. Crystals of p26 were successfully grown at a protein concentration of 8mg/ml in 0.1M cacodylate, 0.7M sodium acetate, pH 6.5, at 4 °C. A sandwich ELISA was developed, using recombinant p26, to detect anti-p26 antibodies in horse sera. The assay successfully identified the 19 positive sera in a blind panel of 30 samples supplied by the National Veterinary Service Labs (Ames, Iowa). Accordingly, the assay appears to meet the criteria required of a commercial diagnostic reagent for determining EIAV infection in horses.

The molecule(s) involved at the axon plasma membrane (axolemma) which causes Schwann cells to proliferate has been investigated by three biochemical techniques: 1) Alkaline extraction of axolemma resulted in recovery of 95% of the mitogenic activity and 50% of the protein in the membrane-bound portion of the axolemma. 2) The axonal mitogen for Schwann cells may be associated with heparan sulfate proteoglycans at the axonal surface. Treatment of axolemma with heparitinase, which cleaves the glycosidic bonds of sulfated glycosaminoglycans, allowed mitogenic activity to be solubilized. 3) Treatment of axolemma with heparin, a highly sulfated glycosaminoglycan analogous to heparan sulfate, resulted in a soluble mitogenic extract which had a higher specific mitogenic activity than the starting material. The results of these biochemical treatments support the model of an axonal mitogen for Schwann cells which is positively charged and bound to the negatively charged portion of heparan sulfate proteoglycans. A monoclonal antibody (1A5-2G3) was raised against the soluble mitogenic heparin extract of axolemma. The monoclonal antibody inhibited the mitogenicity of heparin extract as well as the mitogenicity of the starting axolemmal membrane. Non-specific monoclonal antibodies did not inhibit mitogenicity to as great an extent as 1A5-2G3. Mitogenic heparin extract was incubated with 1A5-2G3-coupled or non-specific antibody-coupled Sepharose. Sepharose coupled with 1A5-2G3 removed significantly more mitogenic activity from the heparin extract than did nonspecific antibodies. Using immunoaffinity techniques with the monoclonal antibody and the soluble heparin extract should permit separation of the axolemmal mitogen from other components of the axon plasma membrane.

Protein subunit structure in the nuclear envelope has not been previously described. Covalent crosslinking of polypeptides provides a method for studying the subunit structure of protein-rich systems. In this study, several polypeptide crosslinking methods have been used to determine associations among polypeptides in isolated chicken erythrocyte nuclear envelope. Interchain associations that exist in the isolated membrane were fixed by crosslinking the polypeptides in the isolated nuclear envelope. Crosslinked and uncrosslinked polypeptides were resolved by electrophoresis in a dissociating detergent system. Each agent caused distinctive alterations in the gel pattern. Certain bands diminished and disappeared while new bands of two or more times their molecular weight appeared in a reciprocal fashion. Most noteworthy was the major peak of approximately 77,000 daltons molecular weight. This peak crosslinked into dimeric species of approximately 160,000 daltons and higher polymeric species. Some components were distinctly unreactive. The preferential crosslinking reactions indicate that the 77,000 dalton species occur in a specific oligomeric arrangement in the native membrane structure.

The lamins are a group of proteins in a residual nuclear envelope fraction derived from the nuclear lamina. The absence of specific functional assays for the two predominant species, lamin A and lamin B, has complicated their biochemical characterization. Initial studies reported in this work involved the enzymic cleavage of isolated lamins A and B and another lamina protein with a molecular weight of 61,000-daltons. The results obtained with each of three enzymes indicated strong similarities between the three nuclear envelope proteins. Further chemical characterization of the lamins has been achieved. Lamins A, B and C have been separated by two-dimensional electrophoresis based on their isoelectric point and molecular weight. Two-dimensional 125 I-tryptic peptide maps were prepared. The results suggest that lamin C is a cleavage product of lamin A, while lamin B has a distinct primary sequence. The topography of the lamins in the isolated nuclear envelope has been examined. Both lamin A and lamin B can be converted to homogeneous polymers by oxidation of intrinsic sulfhydryl groups with o-phenanthroline cupric ion complex. The pattern of polymers formed by oxidative cross-linking was different for lamin A and lamin B. The nature of these homogeneous polymers was investigated further. The crosslinked polymers of lamin A included dimers, trimers, tetramers and larger oligomers. Those that entered the acrylamide gels migrated as expected for their molecular weights. The migration of lamin B oligomers has been found to vary with electrophoretic conditions and this has led to a controversy as to the number of chains in the lamin B oligomers. Three lamin B crosslinked oligomers have been detected and most likely represent a dimer, a trimer and a tetramer. Evidence for this model has been obtained by analysis of cross-linked fragments from a mild trypsin digestion of oxidized lamina proteins. This procedure yields 62,000-, 46,000- and 30,000-dalton fragments of lamin B. Each fragment also occurs as three homotypic oligomers. A further study involved a high resolution two-dimensional gel electrophoretic system for separating the lamina proteins. Using this system a subtype of lamin B has been found in the avian erythrocyte. This subtype, called lamin Bl, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topography.