Meiotic maturation in Xenopus oocytes: a link between the cessation of protein secretion and the polarized disappearance of Golgi apparati

We have studied the relationship between the timing of the late meiotic events that occur during progesterone-induced oocyte maturation, and intracellular protein transport. We have monitored the secretion of chick oviduct proteins from Xenopus laevis oocytes microinjected with polyadenylated mRNA and found that chick ovalbumin and lysozyme are not secreted during the second meiotic metaphase, in contrast to the earlier prophase stage. Maturation had no detectable effect on the glycosylation of ovalbumin, whereas it affected the glycosylation of chick ovomucoid. As maturation proceeded, the Golgi apparati disappeared in a polarized fashion, beginning in the vegetal half. This disappearance coincided temporally and spatially with that of the nuclear envelope. We speculate that Golgi apparatus disappearance and the block in secretion are causally related.     

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.     

Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroides fragilis

Mutants of Bacteroides fragilis sensitive to mitomycin C were isolated after mutagenesis with ethyl methane sulphonate. One mutant (MTC25) was markedly sensitive to mitomycin C but was unaffected as regards UV sensitivity; another mutant (UVS9) was sensitive to UV radiation but was only moderately sensitive to mitomycin C. Caffeine decreased the survival after UV-irradiation of the wild-type, MTC25 and UVS9 strains by the same relative amount. Aerobic liquid holding recovery occurred in each of the three strains. The MTC25 and UVS9 mutants showed reduced host cell phage reactivation. The wild-type, MTC25 and UVS9 strains all showed UV- and H2O2-induced phage reactivation. The physiological characterization of the MTC25 and UVS9 mutants indicates that it is possible to differentiate between mechanisms for the repair of mitomycin C- and UV-induced DNA damage in B. fragilis.     

Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Production of (S)-3-chlorolactaldehyde from (S)-alpha-chlorohydrin by boar spermatozoa and the inhibition of glyceraldehyde 3-phosphate dehydrogenase in vitro

The (S)-isomer of the male antifertility agent alpha-chlorohydrin was metabolized by mature boar spermatozoa in vitro to (S)-3-chlorolactaldehyde. This oxidative process, which did not occur when (R)-alpha-chlorohydrin was offered as a substrate, was catalysed by an NADP+-dependent dehydrogenase that converts glycerol to glyceraldehyde. (S)-3-chlorolactaldehyde, produced by this metabolic reaction or when added to suspensions of boar spermatozoa, was a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase as assessed by the accumulation of fructose 1,6-bisphosphate and the triosephosphates. When glycerol and (S)-alpha-chlorohydrin were added concomitantly to boar spermatozoa in vitro, the presence of glycerol decreased the degree of inhibition of glyceraldehyde 3-phosphate dehydrogenase. Extracts of glyceraldehyde 3-phosphate dehydrogenase that were obtained from boar spermatozoa incubated with (S)-alpha-chlorohydrin or (R,S)-3-chlorolactaldehyde showed significant reductions in their enzymic activity.     

A correlative study of the binding of dexamethasone in hypothalamic blocks in vitro with its ability to inhibit the release of bioactive corticotrophin-releasing factor

Rat hypothalamic blocks incubated in vitro were used to study the characteristics of binding of [3H]dexamethasone and other steroids to cytosolic binding sites. Cytosols prepared following incubation of the tissue with [3H]dexamethasone for 2 h contained specifically bound steroid in amounts that depended upon the concentration of potassium (but not sodium) ions in the extracting buffer. There was an increase in bound [3H]dexamethasone extracted as the potassium ion concentration increased up to 0.1 M, but not beyond. Dexamethasone, when added to hypothalami in vitro caused a biphasic inhibition of bioactive corticotrophin-releasing factor (CRF) release, and the extent of the second phase of inhibition was dose-related. 11-Epicortisol, when added in a 100-fold molar excess over dexamethasone was able to prevent the second phase of inhibition caused by the latter steroid, as in the binding studies it was able to cause a 50% reduction in the binding of [3H]dexamethasone. In the functional studies it was shown that 11-epicortisol was able to "rescue" the tissue from dexamethasone-mediated delayed inhibition of CRF secretion if added to the blocks 30 min (but not later) after the agonistic steroid.