Separation of anomeric aryl glycosides and 1-thioglycosides by chromatography on a cation-exchange resin

Calculations of the electronic distribution in α- and β-D-glucopyranose, β-D-arabinopyranose, and 2-deoxy-β-D-erythro-pentopyranose, as well as in the enediol form of D-erythro-pentulose and its dianion, have been performed by the CNDO/2 method, taking into account the exact geometry of the molecules. The results obtained are discussed in relation to the anomeric effect, dipole moment, ionization constants, and reactivity of the various sugars. The dipole moment of methyl α-D-glucopyranoside was also determined.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.     

Hepatic ligandin subunits and mRNAs during development

Eight-week-old rats had twofold higher hepatic ligandin concentration than 10-day-old animals as determined immunologically and by steroid isomerase and glutathione S-transferase assays. Increased ligandin content was accompanied by parallel increase in subunit synthesis as determined by [³H]leucine incorporation into each subunit relative to incorporation into total cytosolic proteins. The mRNA content for each ligandin subunit was twofold higher in older animals as determined by cell-free in vitro translation followed by immunoprecipitation and dot hybridization using a ligandin cDNA probe. When poly A mRNA from the postmitochondrial fraction of liver from young or old rats was subjected to agarose gel electrophoresis under denaturing conditions and hybridized to ligandin cDNA probe, a single 11 S band was obtained. With RNA from total liver, an additional 13 S band was obtained, suggesting the existence of a precursor form of ligandin mRNA. Since precursor polypeptides were not observed with RNA from total liver in cell-free in vitro translation systems, the precursor form requires processing to the 11 S form before the mRNA becomes functional.     

Competitive binding assays for riboflavin and riboflavin-binding protein

A competitive binding procedure that can be used to determine either riboflavin or riboflavin-binding protein has been developed. Riboflavin-binding protein from chicken egg white binds tightly to DEAE-cellulose while free riboflavin does not. Stock [2-¹⁴C]riboflavin solutions, diluted with varying amounts of a standard unlabeled riboflavin solution or an unknown sample, are mixed with aporiboflavin-binding protein and washed through small DEAE-cellulose columns. The protein-bound riboflavin is batch eluted into scintillation vials, counted, and the unknown samples compared to a standard curve. This is a simple, rapid method for assaying riboflavin by isotope dilution. By a slight modification of the incubation conditions of this procedure, the degree of saturation and amount of riboflavin-binding protein can be determined. Data from both assays can be represented by linear plots in which slopes or intercepts correspond to unknown values. The principles presented here have been extended to the assay of biotin and avidin and should apply to other vitamins and vitamin-binding proteins.     

Formation and stability of the complex formed between human antithrombin-III and thrombin

The inhibition of thrombin by antithrombin-III involves formation of a 1:1 covalent complex between protease and inhibitor and concomitant cleavage of the antithrombin-III peptide chain after Arg-385. The resultant fragment remains connected to the complex via a disulfide bond. This complex spontaneously breaks down into a fragment of approximately 55,000 daltons and smaller peptides. Breakdown is prevented by the presence of hydroxylamine or diisopropylflurophosphate, or by denaturation with urea. It occurs even if the purified complex is treated with diisopropylflurophosphate prior to purification, and can be greatly accelerated by the presence of small amounts of active thrombin. The initial sites of proteolytic attack on the complex are after Arg-13 of the thrombin A chain and Arg-68 of the thrombin B chain. These data indicate that active thrombin can be released from the antithrombin-thrombin complex, and that thrombin becomes more susceptible to proteolytic attack when complexed with antithrombin.     

Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

Increased rate of capping of concanavalin A receptors during early Xenopus development is related to changes in protein and lipid mobility

The mobility characteristics of plasma membrane constituents were studied in dissociated cells from embryos of Xenopus laevis at various stages of development from early blastula until neurulation. An increased rate of fluorescein isothiocyanate-concanavalin A induced patching and capping of Con A-binding proteins during this period of development was correlated with a threefold increase in the lateral mobility of the receptor molecules, as determined by the fluorescent photobleaching recovery (FPR) method, the major change occurring at the onset of gastrulation. Using the same method, it was demonstrated that the lateral mobility of plasma membrane lipids increases twofold during this period of development. The major change being detectable, however, at the late blastula stage. This is in coincidence with the initiation of cell motility in dissociated Xenopus embryo cells. It is concluded that the lateral mobility of membrane proteins and lipids increases significantly during early Xenopus development, but are at least in part subject to different control mechanisms. The results suggest that the initiation of morphogenetic movements is related to changes in the dynamic properties of plasma membrane constituents.     

Regulation of protein synthesis and accumulation during oogenesis in Xenopus laevis

The tissue and developmental distribution of the various myosin subunits has been examined in bovine cardiac muscle. Electrophoretic analysis shows that a myosin light chain found in fetal but not in adult ventricular myosin is very similar and possibly identical to the light chain found in fetal or adult atrial and adult Purkinje fiber myosins. This light chain comigrates on two-dimensional gels with the bovine skeletal muscle embryonic light chain. Thus, this protein appears to be expressed only at early developmental stages in some tissues (cardiac ventricles, skeletal muscle) but at all stages in others (cardiac atria). The heavy chains of these myosins have been examined by one- and two-dimensional polypeptide mapping. The ventricular and Purkinje fiber heavy chains are indistinguishable. They are, however, different from the heavy chain found in cultured skeletal muscle myotubes, in contrast to the situation concerning the embryonic/atrial light chain.     

Syntheses of 2-acetamido-2-deoxy-4-O-βD-galacto-pyranosyl-D-mannose and -D-glucose and of 2-acetamido-2-deoxy-4-O-βD-mannopyranosyl-D-mannose and -D-glucose

2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-mannose (6) and -D-glucose (7) were prepared by addition of nitromethane to 3-O-β-D-galactopyranosyl-D-arabinose, followed by acetylation, ammonolysis, and application of the Nef reaction. Similarly, 2-acetamido-2-deoxy-4-O-β-D-mannopyranosyl-D-mannose (14) and -D-glucose (15) were prepared by the same scheme from 3-O-β-D-mannopyranosyl-D-arabinose. In the two series of experiments, 6 and 14 were the respective major products. Epimerization of the 2-acetamido-2-deoxy-D-mannose residue in 6 and 14 yielded 7 and 15, respectively.     

Decrease of hepatic mono and oligo adenosine diphosphoribose content and augmentation of [14C]ribose incorporation during induction of growth by bovine growth hormone in hypophysectomized rats

Following $\text{in}\text{vivo}$ pulse labeling with [¹⁴C]ribose the specific radioactivities of mono- and polyadenosine diphosphoribose, NAD and adenine nucleotides were determined in livers of hypophysectomized Long Evans rats. These analyses were also performed after induction of growth with four successive daily injections of bovine growth hormone. As a consequence of brief treatment with growth hormone polyadenosine diphosphoribose content markedly diminished whereas its specific radioactivity increased. NAD concentration did not vary but its specific radioactivity increased similarly to that of the homopolymer. The steady state concentration of adenine nucleotides remained unchanged, except for ATP which decreased, and their specific radioactivities uniformly decreased.     

Ribosomal components required for binding protein S1 to the 30 S subunit of Escherichia coli.

30 S subunits of Escherichia coli ribosomes washed with 3 m-NH₄C1 lose proteins S2, S3, S9, S10, S14, S20 and S21, as well as their ability to bind S1 with high affinity (Laughrea and Moore, 1978). Binding activity is restored when the split proteins are added back to the protein-deficient cores. Here we show that, among the split proteins, S9 is by far the most effective in restoring S1 binding capability to 3 m-NH₄Cl cores.     

Formation and stability of cytoplasmic mRNAs during myoblast differentiation: Pulse-chase and density labeling analyses

Stabilities and rates of formation of cytoplasmic mRNAs have been measured quantitatively in cultures of embryonic quail breast myoblasts undergoing differentiation to form muscle fibers. Uridine pulse-chase studies show that dividing myoblasts and differentiated fibers form both short- and long-lived mRNAs. Short-lived mRNAs in myoblasts and fibers have similar half-lives of 2–4 hr, however, long-lived mRNAs have a half-life of 60–100 hr in myoblasts and only 20 hr in fibers. When myoblasts fuse, the formation of long-lived cytoplasmic mRNAs increases at least twofold, and this increased formation together with the cessation of myoblast cell division at fusion is sufficient to account for the four- to fivefold accumulation of long-lived mRNA observed in fibers. These long-lived mRNAs were identified by density labeling cultures with ¹⁵N, ¹³C-nucleotides, chasing with light nucleosides, and then translating the density labeled mRNAs in wheat germ extracts. These experiments show that the contractile protein mRNAs, as well as 60–70 other muscle mRNAs are actively synthesized by muscle fibers and that all of the specific mRNAs detected have half-lives clustering around 20 hr.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

Translation of the mRNAs coding for the major hemolymph proteins of Ceratitis capitata in cell-free system: comparison of the translatable mRNA levels to the respective biosynthetic levels of the proteins in the fat body during development

Four major hemolymph polypeptides (ceratitins) with molecular weights between 8.1 X 10(4) and 8.7 X 10(4) daltons have been identified in the fat body of late Ceratitis capitata larvae. Total fat body RNA from late larvae was translated in reticulocyte lysate, and the predominant in vitro translation products were shown to be the ceratitin precursors. The biosynthesis of these proteins during postembryonic development was studied in both tissue culture and cell-free system. Comparison of the biosynthetic patterns obtained in the two systems suggests a linear relationship between messenger concentration and protein synthesis. Three of these polypeptides show a coordinate pattern of synthesis and are immunologically related. After pupation, all four ceratitins are reabsorbed by the fat body where they accumulate.     

Differential expression of the cellular src gene during vertebrate development

Cellular genes that are homologous to the transforming genes of certain RNA tumor viruses are suspected to play a functional role during normal developmental processes. To investigate this further, we are studying the expression of the cellular homolog of the Rous sarcoma virus transforming gene (c-src) during embryogenesis of fish, frog, and chicken by quantitative determination of the activity of the c-src encoded protein kinase (pp60^c-src). The kinase activity from embryos of fish, frog, and chicken displays the same enzymatic characteristics as the kinase from adult animals: It phosphorylates only tyrosine residues in protein substrates, and its activity is relatively insensitive to inhibition by the diadenosine nucleotide Ap4A. During the course of development, the varying kinase activity level reflects differential expression of the c-src gene product. The kinase activity is low during early development, increases dramatically during organogenesis, and decreases thereafter to the level found in adult animals. The kinase activity displays an organ specificity, with brain showing the highest activity in embryos as well as in adults. Muscle, however, shows high activities during organogenesis, but no or barely detectable activity in adult animals. Our data suggest, therefore, that the c-src gene product plays more of a role in differentiation than in proliferation processes during embryogenesis, and that it may act as a pleiotropic effector.     

The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laevis

Immature, Stage VI oocytes of Xenopus laevis fail to activate (i.e., to propagate a cortical reaction and elevate a fertilization envelope) when pricked or exposed to A23187. We determined the times during maturation when immature oocytes treated with progesterone in vitro developed the capacity to respond to pricking and to ionophore. Responsiveness to ionophore first appears at about 3.5-4.5 hr after progesterone treatment; all oocytes are activated by 8-9 hr after progesterone. The capacity to respond to pricking appears about 1.0-1.5 hr after first signs of ionophore responsiveness. We examined the cortical endoplasmic reticulum (CER) by TEM to determine whether the morphology of this component could be correlated with the development of responsiveness during maturation. Fully mature oocytes exhibit an extensive CER that (1) forms a "shell" around most cortical granules, (2) appears to interconnect cortical granules, and (3) forms junctions with the plasma membrane. The CER-plasma membrane junctions are especially obvious in preparations of isolated cortex. The elaborate CER is not present in immature oocytes. It first appears during maturation of progesterone-treated oocytes at 4.5-5.0 hr, coincident with the time when maturing oocytes develop their responsiveness to ionophore and to pricking. This temporal correlation is consistent with the hypothesis that the CER is one of the components required for regulation of intracellular free calcium in oocytes.     

Adrenocorticotropin administration during early gestation on conceptus development in swine

The purpose of this study was to examine the effect of exogenous adrenocorticotropin (ACTH), administered to gilts during early stages of gestation, upon fetal survival and various maternal and conceptus parameters. Forty-eight gilts of approximately 6-7 months of age were bred by means of artificial insemination after detection of the second estrus and randomly allotted to one of 12 treatment-period groups. Treatment consisted of a daily intramuscular injection of 0, 40 or 80 U.S.P. units of a long acting ACTH preparation for a period of five days. The injection periods were 1-5, 6-10, 11-15 or 16-20 days of gestation with day one corresponding to 48 hours post-estrus detection. All gilts were slaughtered at approximately 37 days of gestation. Forty-two of the 48 inseminated gilts conceived. Conception rate was not different (P>.10) among the 12 treatment-period combinations. Percent fetal survival was greater (P<.09) in gilts receiving 80 U.S.P. units of ACTH (82 +/- 4.3%; X +/- SEM ) than in gilts receiving 40 U.S.P. units of ACTH (68.8 +/- 4.5%). The percent fetal survival in the control group (71.7 +/- 3.9%) was not different (P>.10) from either of the two ACTH treatment groups. A significant (P<.05) treatment by period interaction for percent fetal survival was observed. The lowest percent fetal survival (48.0 +/- 9.0%) was observed in gilts receiving 40 U.S.P. units of ACTH on day 11-15 of gestation. No significant (P>.10) differences were detected among the 12 treatment-period combinations for any of the maternal or conceptus parameters measured.