Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli.

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.     

Fertilizing competency of multiple ovulated eggs in the domestic fowl (Gallus domesticus)

Fertilizing competency of multiple ovulated eggs in the domestic fowl was examined by fertilization in vitro and early development in culture. Normal laying hens (White Leghorn) were treated with 75 IU of PMSG for 7 days followed by injection of anterior pituitary extracts from chickens (CAPE). Ovulation began to occur 7.5 h after injection of CAPE. These hens ovulated 1-7 ova but some premature ovulation of GV stage ova were observed. In vitro fertilization of the multiple ovulated ova was examined by inseminating 10(6)-10(7) sperm onto the germinal disks in m-Ringer's solution. The gamete or zygote nuclei were detected by DNA specific fluorescence using DAPI (4',6'-diamidino-2-phenylindole) in the histological section prepared from the germinal disk. Process of fertilization was examined in the eggs incubated for 4 h after insemination in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Fertilization rate of the total multiple ovulated eggs was 55% (11/20), in which 90% (9/10) and 10% (1/10) in the eggs recovered 7.5-8.5 h and 9.0-9.5 h after CAPE injection were obtained, respectively. Normal pronuclei were formed in five eggs of those recovered 7.5-8.5 h after CAPE injection. Early development after fertilization in vitro was also examined by incubation for 12 h in DMEM + liquid albumen at 41 degrees C under the atmosphere of 5% CO2 in air. Although development in vitro was delayed compared to that in utero condition, normal development was observed in naturally and multiple ovulated eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture

Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Elongation of oligonucleotides in the 3'-direction with activated mononucleotides and their analogs using RNA ligase.

P1-Adenosine 5'-P2-2',3'-ethoxymethylidene nucleosides [A(5')ppN(Em)] from four common nucleosides have been prepared and used for single addition of nucleotides to elongate oligonucleotide chains in the 3'-direction in RNA ligase reaction. U-U-C, T-U-C and A-C-C were used as acceptors. Structural dependence in these acceptors was found to be smaller compared to joining reactions between oligonucleotides. Adenosine analogs including 8-bromo-, 2'-fluoro-, 2'-azido-, 8,2'-O-cyclo-, 8,2'-S-cyclo-adenosine, arabinosyladenine and 2'-deoxyadenosine were added to the 3'-end of A-C-C by adenylation chemically followed by joining with RNA ligase. Symmetrical 5'-pyrophosphates of 8-bromo-, 2'-fluoro- and 2'-azido-adenosine were not recognized as donor substrates.     

Theophylline reduces poly (ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [3H]adenosine-labelled poly(ADP-ribose)molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose)molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecule. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

Pysicochemical studies of taste reception. V. Suppressive effect of salts on sugar response of the frog.

The tast responses of frog to various kinds of sugars were measured quantitatively by use of the glossopharyngeal nerve activity under an appropriate condition where the water response was completely suppressed. The concentration dependences of response of frog tongue to D-fructose, D-glucose, and sucrose were almost the same, D-galactose, however, elicited a much larger response in comparison with the other sugars in the whole range of concentrations examined. The sugar response was suppressed extensively by the presence of small amount of salts in the stimulating sugar solution. The suppressive effects of NaCl, KCl, MgCl2, MgSO4, and K4Fe(CN)6 were examined with a fixed concentration of sugar. The results obtained with these salts, added in various concentrations, fell on a single curve when the data were plotted against the ionic strength in the stimulating solution. The present results were consistent with the notion that the taste receptor potential for salts or acids is attributable to a change in the phase boundary potential at the membrane-solution interface as proposed in the previous papers of this series.     

Alpha reduced nicotinamide adenine dinucleotide-dependent reductase reactions of rat liver microsomes.

The kinetics of alpha-NADH-dichlorophenolindophenol (DCPIP) and alpha-NADH-cytochrome c reductase reactions of rat liver microsomes showed that the reactio ns proceeded by a ping-pong mechanism, and that the oxidation of alpha-NADH was the rate-determining reaction. The DCPIP-reducing activity with alpha-NADH in the presence of ADP was about 1% of that with beta-NADH. ADP inhibited the alpha-NADH-DCPIP reductase reaction in a competitive manner with respect to alpha-NADH and a value of 1.2 mM for the inhibition constant was obtained. ADP also inhibited cytochrome b5 reduction with alpha-NADH. More than 90% of cytochrome b5 was reduced under conditions where 90% of the alpha-NADH-DCPIP reductase activity was suppressed with ADP. The reduction of DCPIP with alpha-NADH preceded that of cytochrome b5, but the reductions partly overlapped. From these results, a diversed electron flow from alpha-NADH to cytochrome b5 and electron sharing between cytochrome b5 and DCPIP were indicated. alpha-NAD+ also inhibited the alpha-NADH-DCPIP reductase reaction. Analyses of the inhibition indicated that two types of alpha-NADH-DCPIP reductase reaction existed, one of which was resistant to alpha-NAD+ inhibition. In contrast to the reoxidation of beta-NADH-reduced cytochrome b5, the process was largely monophasic when cytochrome b5 was reduced with alpha-NADH.