Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein.

To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each strand from DNase I. These sequences are located at the left end of the central palindrome and are shifted four bases relative to one another. On the basis of the DNase protection pattern, we believe this protein to be related to the origin-binding protein defined by Elias et al. (P. Elias, M.E. O'Donnell, E.S. Mocarski, and I.R. Lehman, Proc. Natl. Acad. Sci. 83:6322-6326, 1986). Our DNase I footprint shows both strong and weak areas of protection. The regions strongly protected from DNase I align with the essential contact residues identified by interference footprinting. Methylation interference defines a small binding domain of 8 base pairs: 5'-GTTCGCAC-3'/3'-CAAGCGTG-5'. This recognition sequence contains two inverted 5'-GT(T/G)CG-3' repeats which share a 2-base overlap; thus, the origin-binding protein probably binds to the inverted repeats as a dimer.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.

A new human cyclin, named cyclin E, was isolated by complementation of a triple cln deletion in S. cerevisiae. Cyclin E showed genetic interactions with the CDC28 gene, suggesting that it functioned at START by interacting with the CDC28 protein. Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation. One was CDC2-HS, and the second was the human homolog of Xenopus CDK2. Cyclin E produced in E. coli bound and activated the CDC2 protein in extracts from human G1 cells, and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells. The interactions between cyclin E and CDC2, or CDK2, may be important at the G1 to S transition in human cells.     

Rapamycin resistance tied to defective regulation of p27Kip1.

The potent antiproliferative activity of the macrolide antibiotic rapamycin is known to involve binding of the drug to its cytosolic receptor, FKBP12, and subsequent interaction with targets of rapamycin, resulting in inhibition of p70 S6 kinase (p70S6K). However, the downstream events that lead to inhibition of cell cycle progression remain to be elucidated. The antiproliferative effects of rapamycin are associated with prevention of mitogen-induced downregulation of the cyclin-dependent kinase inhibitor p27Kip1, suggesting that the latter may play an important role in the growth pathway targeted by rapamycin. Murine BC3H1 cells, selected for resistance to growth inhibition by rapamycin, exhibited an intact p70S6K pathway but had abnormally low p27 levels that were no longer responsive to mitogens or rapamycin. Fibroblasts and T lymphocytes from mice with a targeted disruption of the p27Kip1 gene had impaired growth-inhibitory responses to rapamycin. These results suggest that the ability to regulate p27Kip1 levels is important for rapamycin to exert its antiproliferative effects.     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Modification of the hepatotoxicity of D-galactosamine in the rat by cycloheximide.

The effect of cycloheximide (1.5 mg/kg), a potent inhibitor of protein biosynthesis, on D-galastosamine (375 mg/kg)-induced hepatic necrosis and hepatic triglyceride accumulation was studied in rats. Serum transaminase levels, 24 hr after D-galactosamine administration, were significantly reduced in animals treated simultaneously or 4 hr before D-galactosamine with cycloheximide, when compared to animals given D-galactosamine alone. Transaminase levels in rats given cycloheximide 4 hr after D-galactosamine were not reduced. Histological grading of hepatocyte necrosis showed a similar pattern of protection in the pretreated and simultaneously treated groups. Hepatic triglycerides were significantly reduced only in the latter group. Fatality 48 hr after D-galactosamine administration was significantly less common in rats pretreated with cycloheximide when compared to rats given D-galactosamine without cycloheximide, and surviving animals in the cycloheximide pretreated group had a lower serum transaminase level, a lower necrosis score, and a reduced hepatic triglyceride level. These data are consistent with the concept that protein synthesis is important in the pathogenesis of D-galactosamine-induced hepatotoxicity.     

Impact of forest disturbance on the pollen influx in lake sediments during the last century

The pollen accumulation rates of four lakes in different regions of Estonia were estimated in order to study the relationship between pollen influx and the character and intensity of disturbances in the pollen catchment area. The pollen influx data obtained are in accordance with model calculations on the size of the pollen source areas. The influx of arboreal pollen and that of the dominant taxa (mainly Pinus) in the lakes investigated shows that, in the case of small lakes (area 3-6ha) in a forested landscapes, the bulk of the pollen originates from an area within 100-200m around the lake. The distribution patterns of influx from two lakes situated close to each other but at different distances from forest fires show that past disturbances can be reliably detected when the disturbance occurred in the immediate vicinity of the lake and at least 25% of the local pollen source area was involved. In the case of a large lake (137ha) only fires embracing thousands of hectares can be detected in the pollen diagrams.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Replicating viral vectors as HIV vaccines: Summary report from the IAVI-sponsored satellite symposium at the AIDS vaccine 2009 conference

In October 2009, The International AIDS Vaccine Initiative (IAVI) convened a satellite symposium entitled ‘Replicating Viral Vectors for use in AIDS Vaccines’ at the AIDS Vaccine 2009 Conference in Paris. The purpose of the symposium was to gather together researchers, representatives from regulatory agencies, and vaccine developers to discuss issues related to advancement of replication-competent viral vector- based HIV vaccines into clinical trials. The meeting introduced the rationale for accelerating the development of replicating viral vectors for use as AIDS vaccines. It noted that the EMEA recently published draft guidelines that are an important first step in providing guidance for advancing live viral vectors into clinical development. Presentations included case studies and development challenges for viral vector-based vaccine candidates. These product development challenges included cell substrates used for vaccine manufacturing, the testing needed to assess vaccine safety, conducting clinical trials with live vectors, and assessment of vaccination risk versus benefit. More in depth discussion of risk and benefit highlighted the fact that AIDS vaccine efficacy trials must be conducted in the developing world where HIV incidence is greatest and how inequities in global health dramatically influence the political and social environment in developing countries.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.