Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

Infanticide in male laboratory mice: Effects of social status,prior sexual experience,and basis for discrimination between related and unrelated young

Several aspects of infanticide, including effects of social status, prior sexual experience, and the basis for discrimination between related and unrelated young, were examined in Swiss-Webster laboratory mice (Mus musculus). Strange males introduced into the female's cage for 24 h on day 1 postpartum significantly reduced pup survival whereas the introduction of sires did not. Direct observations of infanticide were frequent, and the motor patterns used by males to kill pups are described. Males killed their own offspring when those young were in the nest of a strange female, whereas most males did not kill unrelated young in the nest of a familiar female. Thus, past association with the mother appears to be the single most important factor mediating male discrimination of young. Prior contact with a specific female's urine reduced a male's propensity for subsequently killing her young. The act of copulation itself also reduced infanticide. Infanticidal behaviour was correlated with the male's social status: most dominant males killed unrelated pups, whereas none of the subordinates engaged in infanticide. These results are discussed in terms of the population biology of this species.     

Synthesis of steroids in postimplantation mouse embryos cultured in vitro

Steroid and total lipid synthesis have been assessed in postimplantation stage mouse embryos cultured in vitro from the blastocyst to early somite stage. A large increase in acetate incorporation into these compounds is observed during this period. Cholesterol (60–70%), lanosterol (1–15%), and a fraction containing pregnenolone (0–5%) are the major components of the embryo-associated steroid fraction. When embryos are labeled with [³H]pregnenolone, ³H-labeled progesterone, pregnanedione, and a compound identified as acylpregnenolone are produced and secreted into the medium. Production of progesterone and pregnanedione, but not acylpregnenolone, is severely inhibited by the drug cyanoketone (1 μM). Another drug, SU-10603 (10 μM), severely inhibits pregnanedione production, with only a partial repression of progesterone synthesis, and no effect on acylpregnenolone synthesis. Neither drug affects embryonic development. When embryonic tissues were carefully separated and analyzed for their ability to metabolize [³H]pregnenolone it was observed that all tissues (embryo/yolk sac, yolk sac, and trophoblast) can produce progesterone and acylpregnenolone from pregnenolone. Only embryo/yolk sac and yolk sac, but not trophoblast tissue, can produce pregnanedione. The significance of these observations in relation to metabolic communication between the embryo and its mother is discussed.     

The role of the maturation-promoting factor in controlling protein synthesis in Xenopus oocytes

Oocyte cytosol, containing maturation-promoting factor activity, induces a twofold increase in the rate of protein synthesis as well as inducing germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes. In the current study, it is shown that the cytosol activity responsible for inducing the increase in protein synthesis can be separated from the activity that induces GVBD in recipient oocytes.     

Embryogenesis in the Presence of Blockers of Mechanosensitive Ion Channels

Certain developmental events are thought to be controlled by mechanical tension, but the nature of the transduction mechanism for sensing and responding to tension changes is unknown. A good candidate for such a sensing system would be stretch-activated (SA) ion channels, a type of mechanosensitive (MS) ion channel found in many preparations including the oocytes or embryos of ascidians, fish, and amphibians. To test the hypothesis that SA channel activation is important for early embryogenesis, we treated amphibian and ascidian eggs and embryos with inhibitors of MS ion channels. Xenopus laevis eggs and embryos were treated with gadolinium (Gd³⁺) concentrations up to 100 times the K_d for SA channel inhibition. Boltenia villosa eggs and embryos were exposed to three agents (Gd³⁺, tubocurarine, and gallamine) which are known to block SA channels in other organisms. None of these drugs interfered with morphogenesis in a manner that would suggest SA channel activity is critical to early embryogenesis.     

Changes in cell nuclei during S phase: progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34.

Using multiparameter flow cytometry we have measured the nuclear DNA content of exponentially growing HL-60 cells in conjunction with protein content, nuclear forward light scatter, DNA in situ sensitivity to denaturation, DNA accessibility to 7-aminoactinomycin D (7-AMD), and content of the proliferation-associated proteins: cyclin (PCNA), p105, p34, and Ki-67. Multivariate analysis of the data made it possible to correlate changes in each parameter with the degree of cell advancement through S phase (amount of replicated DNA). A decrease of the protein/DNA ratio, lowered DNA accessibility to 7-AMD, increased sensitivity of DNA to denaturation, and increased ability of isolated nuclei to scatter light all paralleled cell progression through S phase. These changes indicate that during S phase chromatin progressively condenses and suggest that the condensation is associated with the efflux of nonhistone proteins from the nucleus. The increase in the content of the antigen detected by the Ki-67 antibody was observed to exceed the increase in DNA content during S phase and the rate of the Ki-67/DNA increase was higher during the second half of S phase. Thus, this protein appears to be primarily synthesized during S, especially late in S phase, and is degraded in G1. The ratio of cyclin (PCNA)/DNA remained rather constant whereas the contents of p105 and p34 proteins, when expressed per unit of DNA, both decreased during S phase. The data indicate that significant changes in structure and composition of chromatin take place during S phase and suggest that the composition of chromatin associated with the nonreplicated DNA is different compared to chromatin associated with the newly replicated DNA.     

Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel

Summary Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (M _r 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal bythe endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope, 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983.Biochemistry 22:462–470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis.