Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

Cholecystokinin binding and degradation by isolated rat liver cells

The present studies were directed to examine and quantify binding and degradation of radiolabelled cholecystokinin (CCK) peptides by isolated rat liver cells. After incubation with liver cells (4.5 x 10(6) cells/ml) at 14 degrees C, minimal binding (less than 5%) of labelled CCK33 was detected. When labelled nonsulfated (nsCCK8) and sulfated CCK8 (sCCK8) were incubated, 16.2 +/- 1.8% (mean +/- S.E.) and 7.2 +/- 0.1% of 125I-nsCCK8 and 125I-sCCK8, respectively, were bound to the cell fraction. However, no inhibition of binding of either labelled nsCCK8 or sCCK8 was observed when incubated in the presence of excess unlabelled peptide (10 ng-10 micrograms). Preferential binding of labelled sCCK8, the biologically active form of the octapeptide, appeared to be to the nonparenchymal liver cell, rather than the hepatocyte, fraction; when corrected for cell size and protein content, binding of sCCK8 was approximately 15-times greater by the nonparenchymal cell population. When incubated with hepatocytes at 37 degrees C for 60 min, no degradation of labelled sCCK8 was detected by high pressure liquid chromatography. In contrast, progressive degradation of sCCK8 was observed when the peptide was incubated with the nonparenchymal cells. The results of these studies confirm previous observations that CCK33 is not bound by the liver. They further demonstrate that to some degree CCK8 is preferentially bound and degraded by hepatic nonparenchymal cells; however, this binding appears to be noncompetitive and, therefore, probably not receptor-mediated.     

Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Structure-function relationships in the vertebrate U4-U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4-U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4-U6 snRNP assembly or stability. This group includes mutations in both the proposed U4-U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4-U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.     

Chromosome 14 and late-onset familial Alzheimer disease (FAD)

Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analyses were used, in 49 families with a mean age at onset ≥60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependent penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score ≤ –2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P < .02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52, however, was found for a subgroup of families of intermediate age at onset (mean age at onset ≥60 years and <70 years). These results indicate that the chromosome 14 locus is not responsible for Alzheimer disease in most late-onset FAD kindreds but could play a role in a subset of these kindreds.     

Plain tales from the grasslands: extraction, value and utilization of biomass in Royal BardiaNational Park, Nepal

Royal Bardia National Park in Southern Nepal contains a number of internationally endangered wildlife species, and a mix of habitats, including areas of grassland known as phantas. The conservation and current use of these areas is briefly described, including the extraction of grass from inside the National Park by local people living in adjacent villages. The products collected fulfil a range of human needs, both practical and ceremonial. The study attempts to quantify the annual offtake in both material and monetary terms, and comments on the sustainability of the practice. Alternative sources of roofing materials are apparently outside the scope of poorer households. The use of these materials has particular significance given the initiation of a Parks and People Project to implement a Buffer Zone around the National Park and effectively to link conservation and development needs.     

Molecular attenuation of vaccinia virus: mutant generation and animal characterization.

These studies demonstrated that the inbred BALB/c mouse strain can be optimized for the assessment of vaccinia virus virulence, growth, and spread from the site of inoculation and immune protection from a lethal vaccinia virus challenge. The studies established that manipulation of the vaccinia virus genome generated mutants exhibiting a wide range of attenuated phenotypes. The nine NYCBH vaccinia virus mutants had intracranial 50% lethal doses that ranged from 2 to greater than 7 log10 units. The decreased neurovirulence was due to decreased replication in brain tissue. Three mutants had a decreased ability to disseminate to the lungs, brains, livers, and spleens of mice after intranasal infection. One mutant had a decreased transmission from mice infected by tail scarification to naive cage mates. Although the mutants, with one exception, grew to wild-type titers in cell culture, they showed a growth potential on the scarified skin of mice that was dramatically different from that of the wild-type virus. Consequently, all of the mutants had significantly compromised immunogenicities at low virus immunization doses compared with that of the wild-type virus. Conversely, at high immunization doses most mutants could induce an immune response similar to that of the wild-type virus. Three Wyeth vaccine strain mutants were also studied. Whereas the thymidine kinase, ribonucleotide reductase, and hemagglutinin mutants had a reduced virulence (50% lethal dose), only the thymidine kinase mutant retained its immunogenicity.     

Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.     

A View From Both Ends: Shifts in Herbivore Assemblages Impact Top-Down and Bottom-Up Processes on Coral Reefs

Ecosystems - A fundamental goal in ecology is to understand the role of consumers in top-down (TD) and bottom-up (BU) processes that affect the functioning of ecosystems. Consumers ingest organic...     

Wetlands serve as natural sources for improvement of stream ecosystem health in regions affected by acid deposition

For over 40 years, acid deposition has been recognized as a serious international environmental problem, but efforts to restore acidified streams and biota have had limited success. The need to better understand the effects of different sources of acidity on streams has become more pressing with the recent increases in surface water organic acids, or ‘brownification,’ associated with climate change and decreased inorganic acid deposition. Here, we carried out a large scale multi‐seasonal investigation in the Adirondacks, one of the most acid‐impacted regions in the United States, to assess how acid stream producers respond to local and watershed influences and whether these influences can be used in acidification remediation. We explored the pathways of wetland control on aluminum chemistry and diatom taxonomic and functional composition. We demonstrate that streams with larger watershed wetlands have higher organic content, lower concentrations of acidic anions, and lower ratios of inorganic to organic monomeric aluminum, all beneficial for diatom biodiversity and guilds producing high biomass. Although brownification has been viewed as a form of pollution, our results indicate that it may be a stimulating force for biofilm producers with potentially positive consequences for higher trophic levels. Our research also reveals that the mechanism of watershed control of local stream diatom biodiversity through wetland export of organic matter is universal in running waters, operating not only in hard streams, as previously reported, but also in acid streams. Our findings that the negative impacts of acid deposition on Adirondack stream chemistry and biota can be mitigated by wetlands have important implications for biodiversity conservation and stream ecosystem management. Future acidification research should focus on the potential for wetlands to improve stream ecosystem health in acid‐impacted regions and their direct use in stream restoration, for example, through stream rechanneling or wetland construction in appropriate hydrologic settings.