Ontogenesis of TRH mRNA in the rat pancreas

The rapid changes in TRH levels in the rat pancreas during the neonatal period make this organ an interesting model for the study of the regulation of TRH biosynthesis. Pancreatic RNAs were isolated by the guanidinium thiocyanate method and layered onto CsCl cushion. Northern blot preparations were hybridized with 32P labeled TRH cDNA probe. Pancreatic TRH mRNA was first detected in 19-day old fetuses and reached the highest level on day 0, then decreased, being barely detectable 14 days after birth. The neonatal injection of streptozotocin induced a dramatic drop of TRH mRNA levels 24 hours later. This result suggests that the peculiar evolution of TRH level in pancreas is partly due to the evolution of the expression of the TRH gene.     

Human chromosomes. Evidence for autosomal sexual dimorphism.

The karyotypes of 100 males and 100 females, each assembled by the trypsin banding method, are examined in a study designed to investigate sex differences among autosomes. It is shown that female autosomes are consistently longer than those of the males, with respect to both the short and long arm measurements. In addition, discriminant analysis is used to distinguish between the male and female karyotypes. We find that, using autosomal measurements alone, this can be done with a high probability of success.     

Genome-wide analysis of genetic alterations in testicular primary seminoma using high resolution single nucleotide polymorphism arrays

Testicular germ cell tumors (TGCT) represent the most common malignancy among young males. To our knowledge no comprehensive Copy Number Variation (CNVs) studies of TGCT using high-resolution Single Nucleotide Polymorphism (SNP) array have been performed. By a genome-wide analysis of CNV and loss of heterozygosity (LOH) in 25 primary seminomas, we confirmed several previously reported genomic alterations and discovered eight novel genomic alterations including amplifications and homozygous deletions. Moreover, a comparison of genomic alterations of early and late stage seminoma identified CNVs that correlate with progression, which included deletions in chromosomes 4q, 5p, 9q, 13q and 20p and amplifications in chromosomes 9q and 13q. We compared previously perform Affymetrix expression analysis in a subset of samples and found robust correlation between expression and genomic alterations. Furthermore, high correlations (40-75%) were observed between CNV by SNP analysis and quantitative PCR. Our findings may lead to better understanding of TGTC's pathogenesis.     

Evidence for a dinuclear active site in the metallo-beta-lactamase BcII with substoichiometric Co(II). A new model for metal uptake

Metallo-beta-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to beta-lactam antibiotics. Metallo-beta-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-beta-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His(116), His(118), and His(196)) and DCH (Asp(120), Cys(221), and His(263)) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-beta-lactamase turnover.     

Determination of piracetam in human plasma by capillary electrophoresis

A capillary electrophoresis (CE) procedure has been developed for the determination of piracetam in human plasma. Analyses were performed on an uncoated silica capillary using borax buffer modified with the addition of α-cyclodextrin. The detection was UV, operated at 200 nm. The detection limit of the authentic samples was 1 μg/ml. The calibration curve was linear over a range of 4 to 24 μg/ml (r=0.997). Inter-assay R.S.D. was below 9.3%. The described method has been successfully applied to the quantitative determination of piracetam in human plasma and should be useful for clinical and bioavailability investigations.     

The single tryptophan residue of human placental lactogen. Effects of modification and cleavage on biologic activity and protein conformation

In order to define further the chemical features of the human placental lactogen (hPL) molecule responsible for its lactogenic activity, two derivatives of the hormone were prepared by treatment with BNPS-skatole (2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine). At a molar ratio of reagent to hPL of 7:1, a derivative was produced in which the single tryptophan was completely oxidized. At higher ratios, a second derivative was formed in which the peptide chain was cleaved at the tryptophan residue and the two resulting fragments remained bound by the disulfide bond between Cys53 and Cys165. Oxidation of the single tryptophan resulted in reduced immunologic activity, reduced helical content as measured by circular dichroism below 240 nm, and changes in the near-UV circular dichroic spectrum, each indicating a change in the conformation of the hPL molecule. Nevertheless, this derivative retained 20% of its ability to bind to lactogenic receptors and 40 to 50% of its ability to stimulate N-acetyllactosamine synthetase in vitro. Cleavage at the tryptophan was not complete, but the loss of immunologic and biologic activity was equivalent to the degree of cleavage, indicating that the cleaved derivative was completely inactive. In addition, separation of the cleaved fragments from intact hormone followed by recombination did not generate any immunologic or biologic activity. We conclude that the single tryptophan of hPL is not essential for the biologic activity of hPL. It is likely that the reduced activity associated with modification or cleavage at the tryptophan residue is due to changes in the conformation of the molecule.