Modification of testicular and thyroid function by chronic exposure to short photoperiod: a comparison in four rodent species

Exposure of male Syrian hamsters (Mesocricetus auratus) for 10 weeks to short photoperiod (SP) providing 10 hr light: 14 hr darkness (10:14 LD) produced a significant reduction in the weights of the reproductive organs, plasma thyroxine (T4) levels and free T4 index (FT4I) compared to the values of animals exposed to long photoperiod (LP, 14:10 LD). C57bl male house mice (Mus musculus) kept in SP (10:14 LD) had reproductive organ weights equivalent to those of mice kept in long days (14:10 LD) and lower T3 uptake (T3U) values. Male gerbils (Meriones unguiculatus) exposed to 13 weeks of SP (10:14 LD) had lower body weights, testes and seminal vesicle weights and higher T3U values compared to LP (14:10 LD) controls. However, no effect was seen on plasma T4 and triiodothyronine (T3) values nor the FT4I and free T3 index (FT3I). White-footed male mice (Peromyscus leucopus) exposed to SP (8:16 LD) had significantly lower testes and seminal vesicle weights while plasma T4 and T3 levels were unaffected. Snell strain house mice (Mus musculus) exposed to SP (8:16 LD) had normal reproductive organ weights compared to the values of LP-exposed (16:8 LD) control animals. However, there was a significant depression in T3 and in the FT3I in the SP animals.     

Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.     

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm-1 shift from 1306 cm-1 in aqueous solution to 1314 cm-1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm-1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm-1; in water, a strong hydrogen-bonding solvent, 1306 cm-1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm-1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

Quantification of human spermatogenesis: germ cell degeneration during spermatocytogenesis and meiosis in testes from younger and older adult men

Germ cell degeneration during spermatocytogenesis and meiosis was investigated to explain the age-related decline in daily sperm production (DSP). Numbers of Types A-dark, A-pale, and B-spermatogonia, potential daily sperm production per g parenchyma (PDSP) based on type B-spermatogonia, early primary spermatocytes, and late primary spermatocytes, and DSP per g based on early spermatids were determined in 15 men aged 20 to 48 yr (mean +/- SEM, 33 +/- 2 yr) and 15 men aged 52 to 90 yr (65 +/- 3 yr). Testes obtained within 15 h of death (largely due to trauma or heart failure) were perfused vascularly with glutaraldehyde. The number of each cell type per g parenchyma was calculated as the product of the percentage of nuclei in the parenchyma times a correction factor for section thickness and nuclear diameter divided by the volume of a single nucleus of that cell type. Paired testicular weight was lower (p less than 0.01) in older men (33 +/- 3 g) than in the younger men (49 +/- 3 g). Younger and older men had similar numbers of A-dark, A-pale, and B-spermatogonia per g parenchyma. PDSP based on late primary spermatocytes and DSP based on early spermatids were lower (p less than 0.01) in older men than in younger men. In younger men, PDSP was similar (p greater than 0.05) between B-spermatogonia and late primary spermatocytes, whereas DSP measured at the spermatid level was abruptly lower than that estimated from younger cell types. Older men showed reduction in PDSP between early and late primary spermatocytes, with further reduction occurring in DSP at the spermatid level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.     

Organic-solvent water pollution and low birth weight in Michigan.

This pilot study explored the association between a measure of water pollution caused by benzene or chlorinated solvents and the incidence of low birth weights for white residents of Michigan counties. A positive relationship between water pollution by these contaminants and the per cent of low-weight births (less than 2,500 grams, or about 5.5 pounds) resulted despite controls for the incidence of teenaged childbearing, infrequent prenatal care, and mean household income from wages or salaries. Water pollution showed an association with the incidence of low birth weight that was as strong as that between low birth weight and low prenatal care. While correlation cannot prove causation, the finding suggests that impure water may impair fetal growth in Michigan.