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Biomedical subjects

R A Wallace

Publications and source records attributed to R A Wallace.

At least 73 records · Page 4Linked to original sources
PubMed1982

Intracellular glycosylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis.

Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. No apparent role of the rough endoplasmic reticulum in this process was demonstrable. Kinetics of the intracellular translocation of glycosylated vitellogenin indicate that the galactosylated intermediate is secreted more rapidly than the glucosamine-labeled precursor. This was corroborated by measuring the rates of accumulation of various pulse-labeled forms of vitellogenin in the chase medium. In addition, a negligible amount of mannose was incorporated into intracellular or secreted vitellogenin. The antibiotic tunicamycin was shown to inhibit [3H] glucosamine incorporation into microsomal vitellogenin by 70%, without any significant effect on the synthesis of the protein backbone. In addition, nonglycosylated vitellogenin showed normal secretion kinetics. After suitable pretreatment with the antibiotic followed by a labeling period in tunicamycin-free medium, mannose was still not incorporated into vitellogenin, whereas glucosamine behaved in a typical manner. In contrast to this finding, gas-liquid chromatography of the alditol acetate derivatives of the neutral hexoses of vitellogenin showed that mannose was indeed a major component of the vitellogenin oligosaccharide side chain. These preliminary results indicate that the oligosaccharide component of vitellogenin in Xenopus laevis is a "complex" type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to the subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.

Animals↗
PubMed1982

Oocyte growth in the sheepshead minnow: uptake of exogenous proteins by vitellogenic oocytes.

The structure of the vitellogenic follicles of the sheepshead minnow, Cyprinodon variegatus, is described. Follicles enlarge primarily by protein yolk accumulation (vitellogenesis) and subsequently increase in size by hydration. This study uses the electron-dense tracer, horseradish peroxidase, and a larger heterologous protein, Xenopus laevis [3H]vitellogenin, to follow the fate of exogenous proteins from the maternal circulation to yolk spheres of the growing oocyte. Materials appear to leave the perifollicular capillaries via an interendothelial route, traverse the theca and the patent intercellular channels of the follicular epithelium and the pore canals of the vitelline envelope. At the oocyte surface they are incorporated via micropinocytosis and translocated to growing yolk spheres in the peripheral ooplasm. In contrast to other studies on oocyte growth in teleosts which suggest that yolk is an autosynthetic product, this study substantiates the importance of heterosynthetic processes during oocyte growth in C. variegatus.

Animals↗
PubMed1981

Intracellular phosphorylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis.

A procedure was developed for the preparation of rough and smooth microsomes from small quantities of liver obtained from estrogen-stimulated Xenopus laevis females. The separation involves th centrifugation of a postmitochondrial supernatant over a CsCl-containing, discontinuous sucrose gradient. Morphological, biochemical, and enzymatic characterization of these fractions indicates that excellent separation of rough microsomes from smooth microsomes is achieved. In addition, pulse-chase experiments in which (5-min) pulses with [3H]leucine are used demonstrate that rough and smooth microsomes each exhibit the predictable patterns of incorporation characteristic of secretory protein synthesis and intracellular translocation. This procedure was combined with suitable incubation conditions for pulse-chase experiments which demonstrate the subcellular sites of vitellogenin phosphorylation. The data presented indicate that approximately 70% of the phosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of the total incorporated phosphate. This is further supported by the analysis of newly synthesized and assembled [3H,32P]vitellogenin on sodium dodecyl sulfate-polyacrylamide gels and measurements of protein kinase activity in microsomal subfractions.

Animals↗
PubMed1981

Efficient synthesis of 14-hydroxymorphinans from codeine.

Codeine is converted to 7,8-dihydro-14-hydroxynorcodeinone (noroxycodone) in six steps and 52% overall yield of the noroxymorphone in seven steps and 43% overall yield. N-Demethylation and oxidation of codeine afford N-(ethoxycarbonyl)norcodeinone, which is converted to its dienol acetate derivative and oxidized with singlet oxygen to give N-(ethoxycarbonyl)-14-hydroxynorcodeinone in the key step. Hydrogenation of the latter affords N-(ethoxycarbonyl)noroxycodone, which upon acid hydrolysis yields noroxycodone. Alternatively, O-demethylation of N-(ethoxycarbonyl)noroxycodone with boron tribromide and subsequent acid hydrolysis gives noroxymorphone. The results of the singlet oxygen oxidation of the pyrrolidine dienamine derived from N-(ethoxycarbonyl)norcodeinone are also described.

Chemical Phenomena↗
PubMed1981

Full-grown oocytes from Xenopus laevis resume growth when placed in culture.

When most full-grown, follicle cell-invested oocytes from Xenopus laevis are placed in an appropriate culture medium, they resume growth and remain physiologically healthy for at least 2-3 weeks. Rates of growth by full-grown oocytes in vitro generally approximate and can even exceed the most rapid growth rate achieved by vitellogenic oocytes in vivo. Resumption of oocyte growth can be correlated with the loss of investing follicle cells, which under normal conditions appear to interfere with vitellogenin and nutrient access to the oocyte. The final size reached by the oocyte within the ovary is thus not an intrinsic property of the oocyte but is extrinsically imposed by the somatic environment.

Journal Article↗
PubMed1980

Differential postendocytotic compartmentation in Xenopus oocytes is mediated by a specifically bound ligand.

Xenopus laevis oocytes were used as a model system to study the intracellular fate of proteins incorporated by endocytosis. We found that the intracellular stability and compartmentation of proteins incorporated by receptor-mediated endocytosis differed substantially from that of proteins incorporated by nonspecific endocytosis. After its uptake, the specifically sequestered yolk precursor protein vitellogenin was converted to the yolk proteins lipovitellin and phosvitin which were stable with time (up to 13 days in culture). In contrast, nonspecifically incorporated albumin (125I-labeled or 3H-labeled bovine serum albumin) was rapidly degraded. To determine whether the differential stability of these proteins was related to their entry into different postendocytotic compartments, we examined the intracellular transfer pathways taken by these proteins. The transfer of vitellogenin from coated vesicles to the yolk platelets was found to involve a secondary compartment formed by the fusion of the incoming endosomes. This compartment, termed transitional yolk bodies (TYB), underwent a progressive condensation until it attained its terminal density (1.21 g/cm3) after approximately 1.5 hr. The fusion of the TYB with the yolk platelets then occurred coincidentally with the time at which vitellogenin was proteolytically processed into the yolk proteins within the TYB. When the proteolytic cleavage of vitellogenin was blocked there was no fusion of the two compartments. In contrast, we found that albumin incorporated in the absence of vitellogenin was directly transferred from endosomes to yolk platelets without the formation of, or fusion with a secondary compartment. However, when oocytes were exposed simultaneously to albumin and vitellogenin both proteins followed identical routes of compartmentation (that of vitellogenin) with no evidence of direct transfer of either protein to the yolk platelets. These results suggest that the incorporation of a specifically bound ligand can result in the formation of a unique intracellular compartment. Moreover, since yolk platelets were able to fuse only with vesicles lacking occupied receptors (in the case of albumin alone) or with a compartment in which the specific ligand had been proteolytically cleaved and presumably released from its receptor (in the case of VTG), we suggest that occupied receptors can act as a transmembrane signal which directs the postendocytotic compartmentation of proteins.

Animals↗
PubMed1980

Synthesis and antitrypanosomal activity of some bis(4-guanylphenyl) five- and six-membered ring heterocycles.

2,5-Bis(4-guanylphenyl)-1,3-oxazole, 2,5-bis(4-guanylphenyl)-1,3,4-oxadiazole and -1,3,4-thiadiazole, and 3,6-bis(4-guanylphenyl)pyridazine and several of their "cyclic guanyl" analogues have been synthesized. 2,5-Bis(4-guanylphenyl)-1,3-oxazole and -1,3,4-thiadiazole showed good activity, whithout acute toxicity, against Trypanosoma rhodesiense in mice, producing cures at a 3 mg/kg dosage level. This activity is comparable to stilbamidine, hydroxystilbamidine, and pentamidine in this test. In contrast, 2,5-bis(4-guanylphenyl)-1,3,4-oxadiazole shows a sharp reduction in activity in our test system. Generally, the cyclic guanyl analogues exhibit low orders of activity, and toxicity begins to appear at moderate dosage levels. All guanyl and cyclic guanyl compounds were synthesized from bisnitrile precursors by way of imidate ester hydrochlorides in a classical Pinner-type approach.

Animals↗
PubMed1980

Growth of anuran oocytes in serum-supplemented medium.

Appropriately-sized oocytes from Xenopus laevis can be grown in vitro in vitellogenin-containing serum for up to 2 weeks. The source of serum appears to be unimportant. Rates of oocyte growth are somewhat better than those achieved in the presence of vitellogenin alone. Rana pipiens oocytes grow about twice as fast as X. laevis oocytes under identical conditions. The oocyte culture conditions described appear to be applicable to amphibian oocytes in general.

Animals↗
PubMed1980

Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis.

Proteins labeled with either (3)H by reductive methylation or (125)I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and X. laevis serum proteins lacking albumin) were not influenced by the method of labeling. However, (125)I-labeled proteins were apparently degraded at rates far exceeding their (3)H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3-hr incubation, 3-5 times more (125)I-labeled bovine serum albumin and X. laevis serum proteins lacking albumin were degraded than the corresponding (3)H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24-hr incubation, the degradation of (125)I-labeled vitellogenin was 15 times greater than that of [(3)H]vitellogenin labeled in vivo (60% compared to 4%). Moreover, examination of the relative amounts of (3)H- compared to (125)I-labeled bovine serum albumin deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable (3)H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the (3)H-labeled ligands. Passage of dissolved oocytes previously exposed to (125)I-labeled bovine serum albumin (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of bovine serum albumin to intermediate molecular weight components and the presence of a large amount ( approximately 90%) of labeled low molecular weight compounds, which analysis showed to be 72% free iodine. The evolution of either iodotyrosine or free iodine would nevertheless be perceived as protein degradation by most analytical procedures (e.g., acid precipitation or autoradiography). We conclude, therefore, that apparent degradation rates observed for endocytotically incorporated proteins may vary depending on the method used to label the protein and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins.

Animals↗
PubMed1979

The origin of yolk-DNA in Xenopus laevis.

Xenopus laevis serum and plasma was found to contain an average of 25 microgram DNA/ml. Isolated X. laevis oocytes incubated in medium containing 25 microgram DNA/ml labeled with either 125I, 32P or 14C and from three different sources (bovine, E. coli and X. laevis), incorporated the label at an average rate of 0.11 ng.mm-2.hr-1. Sucrose gradient fractionation of oocytes revealed that 40-75% of the acid-precipitable label incorporated was associated with the yolk platelets. Additional incubations of oocytes in unlabeled medium demonstrated that the DNA incorporated into the yolk platelets was undergoing turnover; only 20% of the yolk-associated DNA was still present after a one-week incubation. Our data suggest that yolk-DNA arises by the adventitious uptake of DNA present in the maternal serum by vitellogenic oocytes.

Animals↗
PubMed1979

Calcium-induced dehiscence of cortical granules in Xenopus laevis oocytes.

Microinjection of 0.1 microgram of Ca++ into Xenopus laevis oocytes induces breakdown of the cortical granules. The cortical granules disappeared in both full grown (Stage VI) and small growing (Stage IV) oocytes. Microinjection of Mg++, K+, or Na+ had no effect on cortical granules in either Stage IV or Stage VI oocytes. Small quantities (0.03 microgram) of Ca++ induced dehiscence of the cortical granules only in proximity to the injection site.

Animals↗
PubMed1979

Effects of synchronization and frequency in insemination on fertility.

Fifty-four normally cycling, non-lactating mares were given 2 injections (i.m.) of PGF-2 alpha (10 mg) 14 days apart without regard to stage of the oestrous cycle. At 19 days after the first PGF-2 alpha treatment, a single i.m. injection of either hCG (3300 i.u.) or a GnRH-analogue (500 micrograms) was administered. Each mare was inseminated with 100 X 10(6) motile spermatozoa at one of the following frequencies: once only on Day 20; every other day during oestrus or at least on Days 19 and 21; or daily during oestrus or at least on Days 19, 20, 21 and 22. Eighteen control mares received saline injections on Days 0 and 14, and were inseminated either on the 4th day of oestrus or every other day or daily beginning on the 2nd day of oestrus. More (P greater than 0.05) PGF-2 alpha treated mares displayed their 1st day of oestrus on Days 14 to 20 than control mares (80.6 versus 27.8%). During cycle 1, fewer (P greater than 0.05) treated mares became pregnant compared to controls; 38.9, 25.0 and 66.7% for PGF-2 alpha + hCG, PGF-2 alpha + GnRH-A and control mares, respectively. After three cycles, the pregnancy rates for mares inseminated every other day or daily were higher (P less than 0.05) than mares inseminated only once during oestrus (88.9 and 88.2 versus 64.7%).

Animals↗