Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro

Summary The objective of this in-vitro study was to examine whether the diencephalic floor or the mesenchyme is involved in differentiation of LH cells in the developing rat adenohypophysis. Overall growth of the adenohypophysial tissue was retarded when the adenohypophysial primordium was cultivated after enzymatic removal of the diencephalic floor on days 11.5 and 12.5 of gestation. This malgrowth was more marked when the brain was separated on day 11.5; most expiants retained a simple cystiform structure that consisted of a few layers of undifferentiated cells. Removal of the brain also caused a highly significant decrease (P < 0.001) in the number of immunoreactive LH cells, if it was performed on day 11.5 but not day 12.5. Mesenchyme had little effect on the adenohypophysial growth or the number of immunopositive cells. Cultivation of the adenohypophysial primordium with the diencephalic floor resulted in the appearance of many immunoreactive LH cells. The number of LH cells significantly decreased, however, when the co-cultivated brain completely surrounded the adenohypophysial tissue.These results indicate that in 11.5-day-old fetal rats the diencephalic floor is indispensable for the initial proliferation of adenohypophysial primordial cells and for the early determinating process of LH cells. Once determined, the development of LH cells may proceed without the surrounding tissues. The cytodifferentiation seems to be rather inhibited when in contact with the brain. The significance of the intimate spatial relationship between developing LH cells and the surrounding mesenchyme is also discussed.     

Intra uterine survival and growth of Wistar and Wistar x Brown Norway rat embryos

Embryos of the Wistar strain and its F(1) cross (Wistar females mated with Brown Norway males) of rats were transferred nonsurgically to 48 Wistar, 17 F(1) cross and 20 Wistar-Imamichi recipients. The two types of embryos were transferred together to each recipient to compare the viability of the embryos. Pregnancy rate was 78.8% (67 85 ). The survival rate of fetuses to term was 11.5% (20 174 ) and 25.1% (42 168 ) for the Wistar and F(1) embryos, respectively. Placental weight differed significantly (P<0.05) between embryo types and among recipient types while fetus weight differed (P<0.01) only among recipient types, with a significant interaction between recipient and embryo types (P<0.01). It was concluded that the F(1) embryos (Wistar x Brown Norway) were twice as viable as Wistar embryos under the conditions provided.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

Purification and characterization of an antibacterial factor from snail mucus

The antibacterial factor from the body surface of the African giant snail, Achatina fulica Férussac, was isolated by DEAE-Toyopearl 650M ion exchange chromatography. The isolated preparation exhibited highly positive antibacterial activity both for the Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus and for the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, but it lost such activity when heated at 75 degrees C for 5 min. The antibacterial factor of the snail mucus was a glycoprotein whose molecular weight (MW) was about 160,000. It was composed of two subunits of MW 70,000-80,000.     

Mammalian signal peptidase: partial purification and general characterization of the signal peptidase from microsomal membranes of porcine pancreas

Signal peptidase has been enriched extensively from microsomal membranes of porcine pancreas. Microsomal membranes were washed with 1 M KCl and Brij 35, and then solubilized with 1% Nonidet P-40. The solubilized signal peptidase was purified by DEAE-cellulose chromatography and Sepharose CL-6B filtration. Cleavage of pre-human placental lactogen with the partially purified enzyme gave the mature form, whose NH2-terminus was identified as valine. The signal peptidase is heat-labile and approximately 90% of the enzymatic activity was lost at 60 degrees C within 1 min. The pH optimum of the activity was 7 to 8. Chymostatin and o-phenanthroline at concentrations of 2.5 mM inhibited the signal peptidase activity by 62% and 30%, respectively.     

Food preference,food quality and diets of three herbivorous gastropods (Trochidae: Tegula) in a temperate kelp forest habitat

Summary The relationship between food preference and food quality (i.e. a food's contribution to growth and reproductive development) was examined in the laboratory for 3 species of herbivorous kelp forest gastropods (Tegula). The preference hierarchies of the 3 Tegula for 6 common algal species are the same: giant kelp (Macrocystis) is the most preferred food and brown algae are consumed at higher rates than red algae. Despite their strong preference for Macrocystis, the 3 species had significantly greater growth and/or reproductive development on a mixed-algae diet than on either brown or red algae alone. Laboratory preferences of the snails did not correspond closely with caloric content, estimated availability or quality of the algal species used in this study. However, the 3 Tegula are subject to strong benthic predation and Macrocystis provides an important spatial refuge in nature. The potential role of non-nutritional factors such as predator avoidance on the formation of food preferences is discussed.     

Interaction between 2-chloroadenosine and alpha-adrenoceptors in rat vas deferens

The effect of 2-chloroadenosine (2CA) on the binding of alpha 1- and alpha 2-adrenoceptor ligands in the rat vas deferens was investigated. In homogenates of vas deferens, 2CA (10(5)M) increased 3H-clonidine maximal binding sites from an undetectable level to 0.71 +/- 0.08 pmol/g. wet weight or 10.1 +/- 1.1 fmol/mg protein (N=12). This effect lasted for at least 5 hours after removal of 2CA. Concurrent addition of 1.25 mM theophylline completely abolished the effect of 2CA. A similar effect of 2CA on 3H-clonidine binding was observed following incubation of intact tissues with 2CA prior to homogenization. The effect of 2CA were similar in potency in the homogenate to that in the intact organ, suggesting that 2CA-sensitive sites are located on the outer surface of the plasma membrane. The binding of 3H-prazosin was not influenced by the presence of 10(-5)M 2CA. Contractions of isolated vasa deferentia induced by norepinephrine and phenylephrine were not changed by 10(-5)M 2CA, but the inhibition by clonidine of contractions induced by electric stimulation was enhanced by preincubation for 30 min with 10(-5)M 2CA. The results suggest that 2CA increases the number of available alpha 2-adrenoceptors and this interactions occurs, at least in part, presynaptically.     

Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK.

A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV.     

Genetic basis of the neurovirulence of pseudorabies virus.

Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (US) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessary for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-]) revealed the following. None of the recombinant plaque isolates that were either TK- or which had a deletion in the US was virulent. Not all recombinant plaque isolates which were both TK+ and had an intact US were virulent. These results indicate that both thymidine kinase activity and an intact US were necessary but not sufficient for the expression of virulence. Marker rescue experiments involving cotransfection of the Bartha strain DNA and a restriction fragment spanning the region of the genome that was missing from the Bartha strain resulted in the isolation of virions to which an intact US had been restored. These virions were not virulent but had an improved ability to replicate in the brains of chicks compared with that of the parental nonrescued Bartha strain. Our results show that genes in the US region, which are missing from the Bartha strain, were necessary for virulence but that this strain was also defective in other genes required for the expression of virulence. Thus, the virulence of pseudorabies virus, as measured by intracerebral inoculation into chicks, appears to be controlled multigenically.