Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, FSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of FSV were compared with those of FSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of FSV phages is indistinguishable from that of FSW and (ii) that all viral structural components found in FSW are present in the particles of FSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of FSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the FSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with FSV DNA as a probe revealed that C239 had lost most of the FSV DNA sequence, whereas S-1 had about one copy of the FSV DNA sequence. These results indicate that virulent phage FSV is derived from the lysogenized phage FSW. Therefore, the appearance of FSV can be eliminated by using the prophage-cured derivative of S-1.     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

Human serum catalase decreases endothelial cell injury from hydrogen peroxide.

Serum from normal human subjects contained variable amounts of catalase activity, which was inhibitable by heat, azide, trichloroacetic acid (TCA), or aminotriazole treatment. Serum also decreased hydrogen peroxide (H2O2) concentrations in vitro and H2O2-mediated injury to cultured endothelial cells. By comparison, heat-, azide-, TCA-, or aminotriazole-treated serum neither decreased H2O2 concentrations in vitro nor reduced H2O2-mediated damage to endothelial cells. We conclude that serum catalase activity can alter H2O2-dependent reactions. We speculate that variations in serum catalase activity may alter individual susceptibility to oxidant-mediated vascular disease or be a factor when added to test systems in vitro.     

Decrease of nitrate biosynthesis in scorbutic mutant rats unable to synthesize ascorbic acid

The effect of ascorbic acid deficiency on the urinary excretion of nitrate was investigated using a mutant strain of rats (osteogenic disorder syndrome rats; ODS rats) unable to synthesize ascorbic acid. The amount of urinary nitrate excreted by ODS rats with or without ascorbic acid supplementation were measured before and after the intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). Urinary nitrate excretion increased markedly after LPS injection. Urinary nitrate excretion by ODS rats not supplied with ascorbic acid was significantly less than that of those supplied with ascorbic acid both before and after LPS injection. These results show that ascorbic acid enhances both LPS-stimulated and constitutive nitrate production in vivo.     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Effects of cycloheximide on chromatin condensations and germinal vesicle breakdown (GVBD) of cumulus-enclosed and denuded oocytes in cattle

To determine if newly synthesized protein is imperative for the resumption of meiosis in bovine follicular oocytes collected from small antral follicles, cumulus-enclosed and denuded oocytes were cultured in TCM-199 both with and without various concentrations of the protein synthesis inhibitor, cycloheximide. After 11 h of culture in inhibitor-free medium, all oocytes had undergone germinal vesicle breakdown (GVBD). However, when concentrations of more than 1.0 mug/ml cycloheximide were added to the medium, the meiotic resumption of bovine oocytes was completely blocked. This inhibitory effect of cycloheximide was fully reversible after removal of the inhibitor from maturation media. Germinal vesicle breakdown following removal of cycloheximide occurred twice as fast as in the control medium. Nevertheless, when oocytes were arrested at the germinal vesicle (GV) stage by cycloheximide, a significantly higher proportion of chromatin condensation (40 to 57%) was observed in denuded oocytes than in cumulus-enclosed oocytes (11 to 22%). Thus the cycloheximide treatment could not prevent the chromatin condensation in only denuded oocytes. We conclude that protein synthesis is a prerequisite for GVBD in bovine follicular oocytes and that cumulus cells are responsible for the complementary regulation of the chromatin condensation at the GV stage, regardless of protein synthesis in the oocytes.     

Penetration by bull spermatozoa into the zona pellucida of dead bovine oocytes recovered from frozen-thawed ovaries

The present study was carried out to determine if the zona pellucida of dead bovine oocytes obtained from ovaries stored at -196 degrees C could be used to assess penetrability of capacitated bull spermatozoa. Follicular oocytes were recovered from bovine ovaries which were frozen slowly in a box containing dry ice, plunged into liquid nitrogen, and thawed at 37 degrees C. The dead oocytes were inseminated with various concentrations of spermatozoa preincubated for 0 to 4 h. Sperm penetration rates of the dead oocytes were significantly altered by sperm concentration and preincubation time. Dead and living oocytes matured in vitro (control) gave similar patterns of penetrability based on sperm preincubation time. When sperm concentration was increased, the rate of multiple sperm penetration into the dead oocytes also increased significantly, but the rate of penetration into living oocytes did not alter significantly. All dead oocytes from ovaries stored at -196 degrees C for 1 d to 3 mo were penetrated at similar rates by spermatozoa preincubated for 1-h. Thus, we conclude that dead follicular oocytes recovered from frozen ovaries are useful for the assessment of sperm capacitation and/or the acrosome reaction in cattle.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Measurements of urinary adipic acid and suberic acid using high-performance liquid chromatography

A sensitive and specific method was developed for measuring medium-chain dicarboxylic acids (adipic and suberic acid) in urine. These acids were extracted from urine with diethyl ether and converted into fluorescent derivatives with 9-anthryldiazomethane, which can be separated by high-performance liquid chromatography. The reproducibility was high and the recovery from urine was above 90%. Urinary concentrations of adipic acid in streptozotocin-induced diabetic rats were significantly higher than those in control rats. In diabetic patients, both adipic acid and suberic acid tended to be high, but not significantly. This method should be useful for measuring dicarboxylic acids in urine