Simultaneous determination of creatinine and pseudouridine concentrations in bovine plasma by reversed-phase liquid chromatography with photodiode array detection

A simple, rapid method for the simultaneous determination of creatinine and pseudouridine in bovine plasma is described. Plasma was de-proteinised, concentrated, and chromatographed for 15 min on a C(18) column. Analytes were detected at an optimum wavelength (262 nm) and the internal standard (cimetidine) was detected at 220 nm. The pH of analysis was between 6.5 and 7 where both analytes exist in single chemical forms giving maximum accuracy. Recoveries of both analytes were above 96%. Lowest detectable amounts of creatinine and pseudouridine were 0.28 nmol and 9.0 pmol, and the typical levels detected (+/-SD) were 60 (+/-2.8) and 2.3 (+/-0.10) micromol/L, respectively.     

Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m(3) +/- 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.     

Expression of N-formylated proteins in Escherichia coli

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.     

Analysis of the 3(')-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3'-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed "degradative PCR" and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3'-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S-26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the "glaucescens" clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins

In a calcium-dependent interaction critical for blood coagulation, vitamin K-dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via gamma-carboxyglutamic acid-rich (Gla) domains. Gla domain-mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.     

Immunopathogenesis and immunotherapy in AIDS virus infections

The heterogeneity of HIV and the different human leukocyte antigen (HLA) backgrounds of infected individuals have posed challenges to understanding the pathogenesis of HIV infection. But continuing advances in our knowledge of the role of immune responses in controlling HIV viremia should help to define goals for immune-based therapies and vaccine strategies against AIDS.