The HRAS1 gene cluster: two upstream regions recognizing transcripts and a third encoding a gene with a leucine zipper domain.

We have cloned and characterized a 55-kb region of DNA surrounding HRAS1. It contains a cluster of two, and possibly three, genes associated with CpG islands within the 32 kb immediately upstream of HRAS1. We have sequenced cDNAs representing one of these genes, provisionally designated HRC1. The locus, which is located 29 kb upstream of HRAS1, is divergently transcribed. HRC1 cDNA probe recognizes fragments on Southern blots of DNA from other vertebrate species. In human DNA, multiple homologous fragments are detected in addition to the predicted ones containing HRC1. Therefore, this locus may represent a member of an evolutionarily conserved gene family. HRC1 expression is upregulated with HRAS1 in the EJ bladder carcinoma cell line, suggesting the possibility of coordinate regulation. The deduced translational product of the longest open reading frame (1119 nucleotides, 373 amino acids) predicts a protein with regions rich in glutamine and proline and a region similar to the helix-loop-helix motif adjacent to a carboxy-terminal leucine zipper dimerization motif with four heptad repeats. Alternate splicing of terminal exons occurs, resulting in the truncation of one proline-rich domain and preservation of the leucine zipper. Thus, a biologically important region of chromosome 11p consists of a gene cluster. At least one of these genes, in addition to HRAS1, may be involved in regulation of cell growth or differentiation.     

A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies

Summary A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60° C to 22° C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Addition of Rapamycin to Anti-CD3 Antibody Improves Long-Term Glycaemia Control in Diabetic NOD Mice

Aims/Hypothesis

Non-Fc-binding Anti CD3 antibody has proven successful in reverting diabetes in the non-obese diabetes mouse model of type 1 diabetes and limited efficacy has been observed in human clinical trials. We hypothesized that addition of rapamycin, an mTOR inhibitor capable of inducing operational tolerance in allogeneic bone marrow transplantation, would result in improved diabetes reversal rates and overall glycemia.

Methods

Seventy hyperglycemic non-obese diabetic mice were randomized to either a single injection of anti CD3 alone or a single injection of anti CD3 followed by 14 days of intra-peritoneal rapamycin. Mice were monitored for hyperglycemia and metabolic control.

Results

Mice treated with the combination of anti CD3 and rapamycin had similar rates of diabetes reversal compared to anti CD3 alone (25/35 vs. 22/35). Mice treated with anti CD3 plus rapamycin had a significant improvement in glycemia control as exhibited by lower blood glucose levels in response to an intra-peritoneal glucose challenge; average peak blood glucose levels 30 min post intra-peritoneal injection of 2 gr/kg glucose were 6.9 mmol/L in the anti CD3 plus rapamycin group vs. 10 mmo/L in the anti CD3 alone (P<0.05).

Conclusions/Interpretation

The addition of rapamycin to anti CD3 results in significant improvement in glycaemia control in diabetic NOD mice.     

Missing the target? A critical view on butterfly conservation efforts on calcareous grasslands in south-western Germany

Butterflies are strongly declining on grassland habitats of Central Europe. Therefore, the success of conservation measures on high quality grassland habitats is controversially discussed. We compared the changes in butterfly diversity and community structure on six managed calcareous grasslands with eight unmanaged vineyard fallows. We obtained strong losses of species diversity and remarkable shifts of community compositions on both habitat types. However, the changes on vineyard fallows were only slightly more severe but more stochastic than on the calcareous grasslands. The shifts in community composition with respect to functional species traits were rather similar between the two different grassland types so that complex butterfly communities evolved into generalist-dominated ones. Connectivity was higher among vineyard fallows than among calcareous grasslands. Consequently, conservation measures on calcareous grasslands only partly achieved their goal to maintain the high species diversity and functional complexity still observed in the 1970s. The negative impacts of eutrophication and monotonisation of the landscape as well as climate change are affecting all habitats, independently from management concepts. Therefore, management on conservation sites can buffer against these effects, but is not sufficient for a full compensation.     

ROCK1 phosphorylates and activates zipper-interacting protein kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.     

Genome scan linkage results for longitudinal systolic blood pressure phenotypes in subjects from the Framingham Heart Study

The relationship between elevated blood pressure and cardiovascular and cerebrovascular disease risk is well accepted. Both systolic and diastolic hypertension are associated with this risk increase, but systolic blood pressure appears to be a more important determinant of cardiovascular risk than diastolic blood pressure. Subjects for this study are derived from the Framingham Heart Study data set. Each subject had five records of clinical data of which systolic blood pressure, age, height, gender, weight, and hypertension treatment were selected to characterize the phenotype in this analysis. We modeled systolic blood pressure as a function of age using a mixed modeling methodology that enabled us to characterize the phenotype for each individual as the individual's deviation from the population average rate of change in systolic blood pressure for each year of age while controlling for gender, body mass index, and hypertension treatment. Significant (p = 0.00002) evidence for linkage was found between this normalized phenotype and a region on chromosome 1. Similar linkage results were obtained when we estimated the phenotype while excluding values obtained during hypertension treatment. The use of linear mixed models to define phenotypes is a methodology that allows for the adjustment of the main factor by covariates. Future work should be done in the area of combining this phenotype estimation directly with the linkage analysis so that the error in estimating the phenotype can be properly incorporated into the genetic analysis, which, at present, assumes that the phenotype is measured (or estimated) without error.