Mass spectral analyses of hemoglobin adducts formed after in vitro exposure of erythrocytes to hydroxymethylvinyl ketone

Hydroxymethylvinyl ketone (HMVK) is a reactive oxidation product of 3-butene-1,2-diol, a metabolite of 1,3-butadiene. The potential for HMVK (0.1 and 1mM) to form hemoglobin (Hb) adducts in erythrocytes from Sprague-Dawley rats was investigated at physiological conditions (pH 7.4, 37 degrees C) using electrospray ionization mass spectrometry (ESI/MS). With the 0.1mM HMVK globin samples, the results indicate HMVK adduction on the alpha2, beta2 and beta3 chains. With the 1.0mM HMVK globin samples, adducts were detected on the beta2 and beta3 chains. However, no correlation was observed between incubation time and the extent of adduct formation, and additional adducts were detected when globin samples were fractionated by HPLC before the ESI/MS analyses. For specific localizations of adducts on the globin chains, trypsin digested peptides from the 1mM HMVK globin samples were subjected to liquid chromatography/mass spectrometry analyses. The results, which are consistent with formation of HMVK adducts on several specific peptides within the alpha- and beta-chains, suggest selectivity in the interaction of HMVK with the different cysteine residues in Hb. Because adducts were also detected in peptides containing no cysteine residues and multiple HMVK moieties were detected on some of the cysteine-containing peptides, the results suggest other amino acids may be also reactive with HMVK. Adduct profiles and their relative intensities were consistent between the 1 and 2h samples providing evidence for the HMVK reactions being fast and selective. The finding that fewer peptides were adducted in the 0.1mM HMVK globin samples provides further evidence for selectivity of the HMVK reaction. Collectively, the results show HMVK readily and selectively forms adducts on Hb. Characterization of these adducts will facilitate development of useful biomarkers of exposure to HMVK and its precursor 1,3-butadiene.     

Identification of a novel gene, DZIP (DAZ-interacting protein), that encodes a protein that interacts with DAZ (deleted in azoospermia) and is expressed in embryonic stem cells and germ cells

Evidence from diverse organisms, including humans, suggests that the DAZ (Deleted in Azoospermia) gene and a closely related homolog, DAZL (DAZ-like), are required early in germ cell development to maintain initial germ cell populations. Here we report the identification and characterization of the DZIP (DAZ-Interacting Protein) gene, which encodes at least three different protein isoforms that contain a C2H2 zinc-finger domain. The DZIP gene is expressed predominantly in human embryonic stem cells and fetal and adult germ cells; moreover, two DZIP protein isoforms colocalize with DAZ and/or DAZL proteins in these tissues. Finally, we provide evidence indicating that DZIP may associate with DAZ and its other cofactors in an RNA-binding protein complex that functions in both ES cells and germ cells.     

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.     

Interannual Variation in Timing of Parturition and Growth of Collared Pikas (Ochotona collaris) in the Southwest Yukon

The length of the snow-free season has a significant influenceon reproduction and growth in northern alpine environments,and these life history traits may provide sensitive indicatorsof the responses of organisms to climate change. We examinedgrowth rates and timing of parturition of collared pikas (Ochotonacollaris) from 1995–2002 in the Ruby Range, Yukon Territory,Canada. Growth rates were best described using a Gompertz model,in which the asymptotic mass, determined from the average maleand female weights, was 157 g, the growth rate constant (K)was 0.0557, and the age at inflection (I) was 18.12 days, fora birth weight of 10 g. The maximum growth rate for North Americanpikas (O. collaris and O. princeps) increased with latitude,with maximum growth rates being approximately one-third greaterin northern populations where the snow-free season is less thanthree months long. The mean parturition date varied significantlyamong years from 3 June to 3 July, and delayed parturition wascorrelated with indices of high snow accumulation and, to alesser extent, late spring snowmelt. However, parturition datedid not significantly affect the subsequent over-winter survivalof juveniles in this population, suggesting that pikas are ableto adjust to seasonal uncertainty associated with highly variablespring conditions.     

DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies

The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field. To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51. We show that the loss of gamma-H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis. This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis. In addition, we have observed the presence of gamma-H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA DSBs.     

Feasibility of global gene expression analysis in testicular biopsies from infertile men

Numerous studies have documented the use of microarray analysis to identify patterns of global gene expression that distinguish normal development from that of the diseased state. Yet, there are no reports that compare global gene expression in the fertile and infertile human testis. Here, we report an initial study of global gene expression in testicular biopsies from several men with different infertility phenotypes. We found that microarray analysis of small biopsy samples was suitable for profiling expression of genes known to function in germ cell development and also identified expression of novel genes. Since it is now common for infertile men with spermatogenic failure to use intracytoplasmic sperm injection (ICSI) to achieve biological paternity, we hypothesize that molecular screening of testicular biopsies with microarrays may be suitable: (1) to categorize the molecular phenoytpes of infertile testes in a manner similar to standard morphologic analysis and (2) to initiate larger studies of gene expression in the infertile testes that may identify genetic signatures from biopsies that allow prediction of outcomes.