Heterochromatin markers: A search for heterochromatin specific middle repetitive sequences in Drosophila

We sought for cloned sequences of middle repetitive (MR) complexity that mark obligatory heterochromatic regions. Total genome probes were employed in a differential screening procedure to recover X-specific, Y-specific and autosomal specific heterochromatic sequences. X- and Y-linked sequences were recovered in the same experiment. (Y-linked clones will be described elsewhere). All nine independent, non-identical X-specific clones were found to be partially homologous to one another and to type I rDNA insertion. No other X-specific Bam HI or HindIII clones were found. In situ hybridization to normal and inverted chromosomes revealed extensive homology in the heterochromatin spanning the nucleolus organizer (NOR) and the eu-heterochromatin junction. Eleven clones which are underrepresented in polytene chromosomes were selected in another differential screening. None was autosome-specific. Five were of nucleolar origin. Among them a presumptive type II 28SrDNA insertion sequence was clearly localized within the X-chromosome proximal heterochromatin in addition to the known localization of the X and Y nucleolar organizers. We mapped three clones to major sites on the Y chromosome and to secondary autosomal sites. The results are discussed with regard to the complexity of heterochromatin organization.     

Evaluation of F+ RNA and DNA Coliphages as Source-Specific Indicators of Fecal Contamination in Surface Waters

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.     

Isolation of alcohol dehydrogenases from germinating seeds of pea,broad-bean,lentil and kidney-bean

The maximum of alcohol dehydrogenase (ADH) activity of germinating pea and broad-bean seeds sediments from 40 to 60% ammonium sulfate saturation, from lentil and kidney-bean seeds between 40 and 50%. This operation increased the specific activity of ADH preparations roughly tenfold. Chromatography on DEAE-eellulose and gel filtration increased the activity of the resulting preparation when compared with the initial preparation 178 times with pea, 334 times with broad-bean, 122 times with lentil and 77 times with kidney-bean. The ADHs resemble each other in coenzyme specificity: the reaction rate with NAD is one hundred times greater than with NADP. The substrate specificity is quite wide: besides ethanol, these enzymes oxidize 2-propene-l-ol (actually faster than ethanol), 2-butene-l-ol (at the rate of one half t h a t of ethanol) and butanol (even more slowly). In general, saturated alcohol analogues are oxidized more slowly than unsaturated ones. Methanol is a substrate for the enzym from pea only. The ADHs of the plants studied did not oxidize diols, sugar alcohols and cyclic alcohols. The enzyme from pea has the widest substrate specificity oxidizing isobutanol, phenylalcohol and mercaptoethanol. ADHs, which are widely encountered in plants, resemble each other to a certain degree — they have identical coenzymes, equal K_m values and equal values of the pH optimum, they differ in the purification process and in substrate specificity.     

Activation of pig oocytes using nitric oxide donors

Nitric oxide (NO) plays an important role in intracellular signaling, but its role during the activation of mammalian oocytes is little understood. In our study, in vitro matured pig oocytes were cultured with NO-donors-S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitropruside (SNP). These treatments were able to induce parthenogenetic activation of pig oocytes matured in vitro. The specificity of this effect was confirmed by the activation of oocytes by exogenous endothelial nitric oxide synthase (eNOS) microinjected in the oocyte with its activator calmodulin. Relatively long exposure (10 hr) is needed for activation of pig oocytes with 2.0 mM SNAP. An active NOS is necessary for the NO-dependent activation of pig oocytes because NOS inhibitors L-NMMA or L-NAME are able to inhibit activation of oocytes with NO-donor SNAP. On the basis of our data, we conclude that the NO-dependent activating stimulus seems inadequate because it did not induce the exocytosis of cortical granules. Also, the cleavage of parthenogenetic embryos was very low, and embryos did not develop beyond the stage of eight blastomeres.     

Analytical and biological characterization of supercoiled plasmids purified by various chromatographic techniques

Supercoiled plasmids are an important component of gene-based delivery vehicles. A number of production methods for clinical applications have been developed, each resulting in very high-quality product with low levels of residual contaminants. There is, however, no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity. We have produced two plasmids using four production purification methodologies based on PolyFlo and hydrophobic interaction chromatography (HIC), either alone or in tandem processes. In each case, the product was analyzed using standard molecular biological methods. We also performed a number of biophysical analyses such as dynamic light scattering (DLS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Minimal differences were detected among the preparations based on the more standard molecular biological methods. Some small differences were detected, however, using biophysical techniques, particularly FTIR and DSC, which may reflect small variations in plasmid tertiary structure and thermal stability. Stability after heat exposure at 60 degrees C, exposure to fetal bovine serum and long-term storage at 4 degrees C varied between plasmids. One plasmid showed no difference in stability depending on the production process, but the other showed significant differences. Evaluation in vivo in models for gene immunization and gene therapy showed significant differences in the response depending on the method of purification. Preparations using a tandem process of PolyFlo used in two separation modes provided higher biological activity compared to a tandem HIC/PolyFlo process or either resin used alone in a single column process. These data indicate that the process by which supercoiled plasmids are made can influence plasmid stability and biological activity and emphasize the need for more rigorous methods to evaluate supercoiled plasmids as gene-delivery vehicles.     

The Caenorhabditis elegans UNC-14 RUN domain protein binds to the kinesin-1 and UNC-16 complex and regulates synaptic vesicle localization

Kinesin-1 is a heterotetramer composed of kinesin heavy chain (KHC) and kinesin light chain (KLC). The Caenorhabditis elegans genome has a single KHC, encoded by the unc-116 gene, and two KLCs, encoded by the klc-1 and klc-2 genes. We show here that UNC-116/KHC and KLC-2 form a complex orthologous to conventional kinesin-1. KLC-2 also binds UNC-16, the C. elegans JIP3/JSAP1 JNK-signaling scaffold protein, and the UNC-14 RUN domain protein. The localization of UNC-16 and UNC-14 depends on kinesin-1 (UNC-116 and KLC-2). Furthermore, mutations in unc-16, klc-2, unc-116, and unc-14 all alter the localization of cargos containing synaptic vesicle markers. Double mutant analysis is consistent with these four genes functioning in the same pathway. Our data support a model whereby UNC-16 and UNC-14 function together as kinesin-1 cargos and regulators for the transport or localization of synaptic vesicle components.     

The role of oxidative stress in diabetic complications

The morbidity and mortality associated with diabetes is the result of the myriad complications related to the disease. One of the most explored hypotheses to explain the onset of complications is a hyperglycemia-induced increase in oxidative stress. Reactive oxygen species (ROS) are produced by oxidative phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase (NADPH), xanthine oxidase, the uncoupling of lipoxygenases, cytochrome P450 monooxygenases, and glucose autoxidation. Once formed, ROS deplete antioxidant defenses, rendering the affected cells and tissues more susceptible to oxidative damage. Lipid, DNA, and protein are the cellular targets for oxidation, leading to changes in cellular structure and function. Recent evidence suggests ROS are also important as second messengers in the regulation of intracellular signaling pathways and, ultimately, gene expression. This review explores the production of ROS and the propagation and consequences of oxidative stress in diabetes.     

Combined immunization with fusion genes of mutated E7 gene of human papillomavirus type 16 did not enhance antitumor effect

BACKGROUND: The E7 oncoprotein of human papillomavirus type 16 (HPV16) is frequently used as a model tumor-associated antigen. Its immunogenicity has been substantially enhanced by fusion with several proteins of various origins and functions. Different mechanisms have been responsible for increased vaccination efficacy of fusion proteins. METHODS AND RESULTS: We linked E7 and its mutated form (E7GGG) with the mouse heat-shock protein 70.1 (HSP70.1). Enhanced immunogenicity of both fusion genes administered via a gene gun was demonstrated by protection of C57BL/6 mice against oncogenic MHC class I positive TC-1 cells producing the HPV16 E7 oncoprotein but not against the MHC class I negative TC-1/A9 subline. To assess if the efficacy of E7-based DNA vaccines could be increased by combination of various fusion genes, we combined the HSP70.1 fusion genes (i.e. E7HSP or E7GGGHSP) with the fusion construct linking E7GGG with targeting signals of lysosome-associated membrane protein 1 (Sig/E7GGG/LAMP-1). Treatment of mice 4 days after TC-1 cell inoculation showed moderately higher immunization potency of HSP70.1 fusion genes in comparison with the Sig/E7GGG/LAMP-1 gene. Any combination of two fusion genes given in the same gene gun shot neither was more effective compared with single genes nor protected mice against TC-1/A9 cells. As fusion of E7GGG with E. coli glucuronidase (E7GGG.GUS) had been previously proven to provide partial protection from TC-1/A9-induced tumors, we also combined E7GGGHSP with E7GGG.GUS. The genes were inoculated either in mix in two gene gun shots or separately each gene in one shot into opposite sides of the abdomen. Neither mode of combined immunization induced higher protection than E7GGG.GUS alone. However, doubling the DNA dose considerably enhanced the antitumor efficacy of E7GGG.GUS. CONCLUSIONS: We constructed highly immunogenic fusions of HPV16 E7 and E7GGG with mouse HSP70.1. Furthermore, we substantially enhanced protection against TC-1/A9 cells with downregulated MHC class I expression by doubling the pBSC/E7GGG.GUS dose, but we failed to demonstrate a beneficial effect of any combination of two fusion genes with different mechanisms causing enhancement of HPV16 E7 immunogenicity.     

PIML: the Pathogen Information Markup Language

MOTIVATION: A vast amount of information about human, animal and plant pathogens has been acquired, stored and displayed in varied formats through different resources, both electronically and otherwise. However, there is no community standard format for organizing this information or agreement on machine-readable format(s) for data exchange, thereby hampering interoperation efforts across information systems harboring such infectious disease data. RESULTS: The Pathogen Information Markup Language (PIML) is a free, open, XML-based format for representing pathogen information. XSLT-based visual presentations of valid PIML documents were developed and can be accessed through the PathInfo website or as part of the interoperable web services federation known as ToolBus/PathPort. Currently, detailed PIML documents are available for 21 pathogens deemed of high priority with regard to public health and national biological defense. A dynamic query system allows simple queries as well as comparisons among these pathogens. Continuing efforts are being taken to include other groups' supporting PIML and to develop more PIML documents. AVAILABILITY: All the PIML-related information is accessible from http://www.vbi.vt.edu/pathport/pathinfo/