Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 ) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H₂O and D₂O were used as solvent. For both the addition and the elimination of SO₃²⁻ values of k₂^H/k₂^D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 , pH 7.2, are 10.2 and 1.3 ⁻¹, respectively.     

The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster.

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.     

Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue

Summary The deffects of a purified homologue of tunicamycin (B₂-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6–8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B₂-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B₂-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B₂-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - MF mouse fibroblasts from Balb/c mouse embryos - 3T3 Balb/3T3 mouse fibroblastic line - SV40 Simian virus 40 - PY polyoma virus - TLC thin layer chromatography     

甜菊不同叶龄细胞结构及其甜菊糖甙含量分布的研究

本文报道甜菊(Stevia rebaudiana Bertoni)不同叶龄细胞结构与甜菊糖苷含量分布。应用电镜技术观察表明,现蕾期成叶细胞内具有内含物丰富的巨大液泡,这些内含物呈大小不一的颗粒或小泡。应用差速离心法,对甜菊成叶的叶肉细胞进行亚细胞分离,并对各部分进行甜菊糖苷的提取与微量测定。结果表明,甜菊糖苷主要存在于12000g的上清液(这部分主要包括液泡内含物和可溶性细胞质)。结合细胞结构和细胞化学研究结果,表明细胞质是合成UDPG的主要场所,在甜菊糖苷合成中具有重要作用。对不同叶龄叶片甜菊糖苷测定表明,现蕾期成叶的甜菊糖苷含量最高。从甜菊不同叶龄细胞结构和甜菊糖苷含量测定结果,现蕾期是甜菊叶片收割的最适时期。     

Clinical and Serological Response after Experimental Inoculation with Babesia Divergens of Newborn Calves with and without Maternal Antibodies

The influence of maternal antibodies on clinical and serological response after experimental inoculation with Babesia divergens of newborn calves was studied. Five calves, born to dams seropositive for B.divergens, (Group 1) had specific maternal antibodies when tested 12 h after their first feeding of colostrum. At that point they were inoculated i.v. with B.divergens infected erythrocytes. Five other calves, born to dams seronegative for B.divergens, (Group 2) had no Babesia specific maternal antibodies when inoculated at the same age. Babesia divergens organisms were demonstrated in blood smears from calves in both groups at some point 5 to 10 days p.i. All calves in both groups had B.divergens specific IgM antibodies at 7 to 17 days p.i. as shown by a modified IF-test. Specific IgG antibodies, transferred by colostrum, were found in all calves of Group 1 before inoculation of B.divergens. The IgG titre of these animals increased by a doubling dilution step at 11–25 days p.i. Among calves of Group 2 specific IgG antibodies were found at first between day 9 and 15 p.i. Both IgM and IgG antibody titres had to be investigated since demonstrated IgG antibodies can originate both from maternally transferred antibodies and from actively produced antibodies after an infection. There was no difference in clinical parameters; parasitaemia, PCV, Hb, and rectal temperature between the groups. This experiment gives evidence that there can be a resistance to bovine babesiosis in newborn calves independent of maternal antibodies.     

Effect of rhodium (III) complex on experimental carcinogenesis in the livers of rats treated with thioacetamide

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats. The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration. The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.     

Epinemin: a new protein associated with vimentin filaments in non-neural cells

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."     

Enhancement of the repair of carcinogen-induced DNA damage in the hamster pancreas by dietary selenium

We measured single strand breaks (SSB) in pancreas DNA produced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters fed purified diets containing added sodium selenite (Se) at 0.0, 0.1 and 5.0 ppm. There were fewer SSB in those given the 5.0 ppm Se diet throughout the experiment. One hour after dosing with BOP (20 mg/kg), there were 2.26 ± 0.47, 2.83 ± 0.43 and 1.74 ± 0.43 SSB per 10⁸ daltons (mean ± S.E.M.) respectively in the three groups. The SSB were repaired faster in the 5.0 ppm Se-fed group. The approximate half-lives of the SSB were 33, 30 and 8 days, respectively. In the hamsters fed 5.0 ppm Se there was a small, statistically significant increase in pancreatic DNA synthesis. Autoradiographic analysis indicated that this was repair synthesis. In a second experiment, hamsters were fed one of the three diets prior to and for 2 days after administration of a single dose of BOP (20 mg/kg). They were then fed the 5.0 ppm Se diet for 5 days. The number of SSB was compared with those in hamsters fed their original diet for 7 days after BOP dosing. There was a statistically significant difference in the number of SSB in the hamsters fed 0.1 ppm Se before and for 2 days after BOP. In these hamsters there were 1.21 ± 0.24 SSB per 10⁸ daltons compared with 3.19 ± 0.4 (mean ± S.E.M.). These results suggest high levels of dietary Se stimulate the repair of carcinogen-induced DNA damage.     

The in vitro synthesis and characteristics of ribosomal RNA in imaginal discs of Drosophila melanogaster

Summary Late third instar imaginal discs of Drosophila melanogaster cultured in vitro in Robb's tissue culture medium synthesize 38S, 28S and 18S ribosomal RNAs which are qualitatively indistinguishable from their in vivo synthesized counterparts (Fig. 1). As found in other insect systems, the 38S molecule appears to be the precursor for both the 28S and 18S rRNAs (Figs. 2, 3 and 4). The 28S rRNA and a portion of the 38S pre-rRNA shift in sedimentation value upon exposure to heat or dimethylsulfoxide (Figs. 5 and 8). Studies of the thermal denaturations of these molecules (Figs. 6, 7 and 9) indicate the existence of a single class of 28S rRNA, but three classes of 38S pre-rRNAs. The addition of -ecdysone to the in vitro culture medium stimulates the net amount of rRNA synthesized, increases the rate of processing of the 38S precursor and increases the relative amount of 18S material produced (Figs. 10 and 12).This work was supported in part by grants from the National Science Foundation (GB-8176) and from the Atomic Energy Commission (AT-04-3-34).Predoctoral Trainees, PHS Training Grant No. 2-Tl-GM367 from Research Training Grants Branch, National Institute of General Medical Sciences.1 For purposes of simplification we shall refer to the rRNA molecules of D. melanogaster as being 38S, 30S, 28S and 18S; however, it should be noted that these values are approximate (see Hastings and Kirby, 1966; Greenberg, 1969; Tartof and Perry, 1970).