Effect of centchroman on tubal transport and preimplantation embryonic development in rats

A single oral administration of centchroman (1.25 mg/kg) to adult female rats within 24 h of mating induced slight acceleration in the rate of transport of embryos through the oviducts. The compound did not seem to produce any deleterious effect on preimplantation embryonic development since well organized and apparently normal embryos were collected from the genital tract up to Day 12 of pregnancy. The recovery rate of embryos from centchroman-treated rats was, however, significantly reduced after Day 4 of pregnancy. There was some stimulation in the rate of cleavage of embryos and morula to blastocyst transformation, but retardation in the shedding of the zona pellucida. The rate of blastocyst formation was not altered when 6-8-cell embryos collected from the oviducts of control rats were transferred to the uteri of control or centchroman-treated females. A delay in zona shedding was observed in the centchroman-treated recipients.     

Protection from quinidine or physostigmine against in vitro inhibition by sarin of acetylcholinesterase activity

We have studied the relative effectiveness of quinidine and physostigmine in protecting against the inhibition of acetylcholinesterase (AChE) by sarin, an organophosphate (OP) compound. The protective effects of these compounds were studied in vitro in both synaptosomal and soluble samples obtained from various regions of sarin-administered or control isolated, perfused canine brain. Although AChE activities in the sarin-administered brain were substantially lower than in the control brain, we observed regional differences in the AChE activity in both. The AChE in the control brain and the AChE remaining in sarin-administered brain had different susceptibilities to inhibition from OP compounds in vitro and, therefore, have different properties. Quinidine partially protected AChE from the inhibitory effects of sarin in vitro possibly by altering the sarin binding sites. Addition of sarin to physostigmine-treated control brain samples allowed partial recovery of the AChE activity. The protective effects of quinidine or physostigmine were lost when samples from sarin-administered brain were treated in vitro with these compounds and then again exposed to sarin. Therefore, both quinidine and physostigmine provided partial protection against the inhibitory effects of sarin in vitro if they were added prior to sarin.     

Transposon mutagenesis in Azospirillum brasilense: isolation of auxotrophic and Nif- mutants and molecular cloning of the mutagenized nifDNA

Summary We report the successful mutagenesis of Azospirillum brasilense 29710 Rif Sm with transposon Tn5. The narrow host-range plasmid pGS9 (p15A replicon), which possesses broad host-range N-type transfer genes, was used as the suicide vehicle to deliver Tn5 in Azospirillum. Out of 900 colonies tested, 0.8% proved to be auxotrophic. One mutant altered in indoleacetic acid (auxin) biosynthesis was isolated and, in addition, three mutants completely defective in nitrogen fixation (nif) were obtained. All the mutants tested contained a single copy of Tn5 integrated randomly in the genome. The Tn5-mutagenized EcoRI fragments were cloned from the three Nif^- mutants. Physical analysis of cloned DNA showed that Tn5 was present on a different EcoRI fragment in each case, ranging in size from 15–17 kb. The nitrogenase structural genes (nifHDK) in A. brasilense 29710 Rif Sm were localized on a 6.7 kb EcoRI fragment. We found that Tn5 is not inserted in the nifHDK genes in the Nif^- mutants reported here. Site-directed mutagenesis using the cloned, Tn5-containing DNA from mutant Nif27(pMS188), produced a large number of Nif^- transconjugants of the A. brasilense 29710 Rif wild-type strain, showing the linkage between Tn5 insertion and the Nif^- phenotype. This is the first time that transposon-mutagenized auxotrophic, Nif^- and other mutants have been available for genetic analysis in Azospirillum. This should greatly facilitate the cloning and mapping of genes involved in nitrogen fixation as well as in many other phenotypic characteristics of Azospirillum.     

A Possible Role for Indoleacetic Acid, Low Temperature, and Phospholipid Metabolism in the Induction of GA(3) Responsiveness in GA(3) Insensitive (Rht3-Containing) Dwarf Wheat Aleurone

Preincubation of dwarf, Rht3-containing deembryonated seed for 4 hours in 342 nanomolar indoleacetic acid (IAA) induced maximum sensitivity to GA₃. In addition, the 4-hour IAA pretreatment caused a 2-fold increase in total phospholipids which coincided identically on a temporal basis with the induced GA₃ sensitivity. Changes in absolute levels of individual phospholipids and their acyl groups were recorded and compared with the changes observed in several Rht-containing aleurone tissues which were induced to develop GA₃ sensitivity by exposure to low temperature (5°C). Several distinct similarities between all tissues were recorded as they develop GA₃ sensitivity. One parameter, the percentage phospholipid composition, was quite similar in all tissues after they had become maximally sensitive to GA₃, suggesting that there is at least one membrane phospholipid composition which is particularly responsive to GA₃. The results indicate that (a) the basis of the GA₃ insensitivity of the Rht mutation resides in an aberrant phospholipid/fatty acid composition and/or metabolism; (b) exposure to low temperature (5°C) for 20 hours or longer, or 342 nanomolar IAA for 4 hours or longer reverses or corrects the genetic lesion, enabling the tissue to adopt a GA₃ responsive membrane composition. Finally, an hypothesis is discussed which indicates that IAA may play a controlling role in the mobilization of endospermal reserves, at least in Rht3-containing wheat aleurone.     

Lymphopoiesis in the nude fetal thymus following sympathectomy

This study was carried out to examine the innervation of the nude fetal thymus during ontogeny and to see if lymphopoietic activity would occur within these thymic lobes in the absence of sympathetic neuronal input. Fetal thymic rudiments from nu/nu mice were removed and examined for galoxylic acid-induced histofluorescence to detect the catecholaminergic nerves. Some of these lobes were organ cultured for 5 to 7 days in the presence of deoxyguanosine to eliminate any existing lymphoid cells within the rudiments. Such "nonlymphoid" thymic rudiments were implanted into the anterior eye chambers of syngenic BALB/c mice (heterozygous) from which cervical sympathetic ganglia and part of the sympathetic chain had been surgically removed (right side) one week earlier. The left side was only sham operated. The thymic implants were allowed to grow for up to 21 days on both sides; they were then removed and examined by histofluorescence, immunofluorescence, and light microscopy. The results indicate for the first time that the nude fetal thymus is innervated by sympathetic nerves and that following sympathectomy the nude thymus is able to sustain lymphopoietic activity and generate lymphoid cells which have characteristics present on thymocytes during in vivo development in normal mice, such as binding to peanut agglutinin and expression of Thy-1 antigen. The relationship between the presence of sympathetic inhibitory influence and the thymic atrophy seen in the nude mice during ontogeny, is being investigated.     

Differential induction of chromosome puffs in two cell types of Melanagromyza obtusa

The patterns of puffing activity in polytene nuclei of salivary gland (SG) and midgut (MG) tissues of Melanagromyza obtusa have been studied after heat shock (HS), 2-4-dinitrophenol (DNP) or benzamide treatment. This study has revealed that HS and DNP treatments induced the same set of puffs but in a tissue-specific pattern. Benzamide treatment was found ineffective in inducing puffing activity. Some HS genes were also found to be more or less active during normal development, indicating some function in the normal metabolism of the cells.     

Yeast fatty acid synthase: structure to function relationship

The yeast fatty acid synthase is a multifunctional enzyme composed of two nonidentical subunits in an alpha 6 beta 6 complex that is active in synthesizing fatty acids. The seven catalytic activities required for fatty acid synthesis are divided between the alpha and beta subunits such that the alpha 6 beta 6 complex has six complements of each activity. It has been proposed that these are organized into six centers for fatty acid synthesis. There are different opinions regarding the operation of these centers in the alpha 6 beta 6 complex, on view being that they are functionally independent and the other proposes half-sites activity for the complex. We have attempted to distinguish between these proposals by the most direct method of active site titration, i.e., quantitation of fatty acyl product in the absence of turnover. This was accomplished by using p-nitrophenyl thioacetate and thiophenyl malonate (in place of the coenzyme A analogues) as substrates along with NADPH, thereby depriving the yeast synthase of coenzyme A required to release product as fatty acyl coenzyme A. The amount of fatty acyl product formed was quantitated by gas-liquid chromatography, as well as by direct estimation of radioactivity in the product when p-nitrophenyl thio [1-14C] acetate was used as a substrate. In both cases, a stoichiometry of close to six was found for mole of fatty acid synthesized per mole of alpha 6 beta 6 complex. This indicates that there are six functional centers for fatty acid synthesis in the multifunctional yeast alpha 6 beta 6 fatty acid synthase and that these centers operate independently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced virulence of Yersinia enterocolitica by copper-induced injury.

A sublethal concentration of copper (0.75 mg/liter) caused substantial injury (87 to 95%) of Yersinia enterocolitica serotype O:8 cells in 72 h at 4 degrees C without producing extensive cell death. Copper-injured cells had a higher 50% lethal dose in mice (2,700 CFU) than uninjured cells (150 CFU). This reduced virulence correlated with more rapid clearance of the injured cells from the blood of mice after intravenous inoculation. A possible role of the liver in this process was shown by significant cell accumulation in mouse livers when copper-injured Y. enterocolitica cells were administered, compared with uninjured bacteria. In vitro studies with isolated mouse liver membranes showed higher titers of aggregation with copper-injured cells than control cells. The in vitro aggregation reaction and blood clearance activity in vivo were abolished by sugars that are known to interact with a hepatic lectin. Our data suggest that copper-induced injury reduces the virulence of Y. enterocolitica and that the liver may be involved in nonimmune rapid clearance of the injured cells, probably by interaction with a hepatic lectin(s).     

Poly ADP-ribosylation of histones in tumor promoter phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts C3H10T1/2

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the poly ADP-ribosylation of acid extractable (0.2N H2SO4) nuclear proteins in mouse embryo fibroblasts C3H10T1/2. Catalase suppresses the reaction by approximately 50%. Polyacrylamide gel electrophoresis reveals that the core histones H2B, A24 and H3d serve as major poly ADP-ribose acceptors. Smaller amounts of poly ADP-ribose are associated with histones H2A/H3 and H1. Poly ADP-ribosylation of histones may change the nucleosomal structure and function and play a role in PMA induced modulation of gene expression in promotion.