Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of physico-chemical factors on anion-sensitive ATPase activity

Effects of temperature, pH and anions on the ATPase activity of submitochondrial particles of rat liver, rat heart, mouse liver, of red blood cell membranes, and of soluble enzyme of rat liver, mouse liver mitochondria were studied. The temperature relationships of membrane-bound and soluble ATPases have the breaks at 18-21 degrees C and 30-32 degrees C. These breaks were not shifted by sulfite, thiocyanate, methanol, glycerol and GTP. The pH changes from 6.0 to 8.5 produced no effect on the temperature relationships of ATPase activities but, strongly influenced the rate of ATPase reaction. The conformity between the obtained data and earlier proposed mechanism of anion control over anion-sensitive ATPase activity was discussed.     

Mutants of Chinese hamster cells deficient in thymidylate synthetase

Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the "FAT" medium increased more than 100-fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5-7 days after mutagen treatment. The recovery of FAT-resistant colonies in the selective medium was not affected by the presence of wild-type cells at a density below 9,000 cells per cm2. All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild-type activity in three prototrophic revertants, as measured by whole-cell and cell-free enzyme assays. The apparent Michaelis-Menten constant (Km) for deoxyuridine-5'-monophosphate and inhibition constant (Ki) for 5-fluoro-deoxyuridine-5'-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6-3H]-2'-deoxyuridine 5'-monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT-resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis.     

Formation and Clearance of Norepinephrine Glycol Metabolites in Mouse Brain

To determine the degree of conversion of 3,4-dihydroxyphenylethyleneglycol (DHPG) to 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and the amount of DHPG eliminated unchanged from the brain, we have examined the kinetics of formation and disappearance of mouse brain MHPG and DHPG following clorgyline (10 mg/kg, i.p.) and/or tropolone (75 mg/kg, i.p.) treatment. During the first 10 min after tropolone, brain DHPG levels accumulated linearly at a rate of 1,300 pmol/g/h, whereas MHPG disappeared exponentially at a rate of 411 pmol/g/h. Following clorgyline administration, brain DHPG declined exponentially at a rate of 1,240 pmol/g/h. In contrast, the elimination of MHPG became a first-order process only when catechol-O-methyltransferase (COMT) was also inhibited in addition to monoamine oxidase. Thus, combined clorgyline and tropolone treatment resulted in an exponential decline of MHPG levels at a rate of 524 pmol/g/h, whereas DHPG levels were slightly but significantly elevated compared to control values. When the animals were treated with pargyline (75 mg/kg, i.p.) in combination with clorgyline and tropolone, brain DHPG and MHPG disappeared at rates of 40 and 660 pmol/g/h, respectively. The above observations suggest that mouse brain DHPG is cleared primarily through O-methylation with minimal direct elimination from brain. Assuming the disposition and clearance of norepinephrine metabolites are similar in mouse and human brain, peripherally measured DHPG in humans is likely derived principally from extracerebral sources and reflects peripheral sympathetic function.     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

A simple purification of calmodulin by reversed phase chromatography with hydrophobic polymer 3520

A new adsorption chromatography procedure for the purification of calmodulin from bovine brain was developed using polymeric adsorbent 3520. Calmodulin was first isolated by DEAE-Cellulose column chromatography and further purified to apparent homogeneity following elution with 50% ethanol from the adsorbent column. Polyacrylamide gel electrophoresis showed one band either in the presence of Ca2+ or EGTA. The polymeric adsorbent 3520 is a non-polar polymer lacking exchangeable groups. The selective adsorption of calmodulin is based on hydrophobic interaction within the matrix, and is Ca2+ independent. Neither high salt (0.5 M NaC1) nor EGTA (5 mM) was able to elute the CaM from the adsorption column whereas ethanol (50%) eluted it completely. This method is simple to use and it provides highly purified calmodulin with high yield.     

二十三种药用种子(或果实)中油的化学组成

本文报道了二十三种药用种子(或果实)的含油率和油的化学组成成分,它们分属于二十个科二十二个属中。本文所发表的大部分资料尚未见国内外文献的报道。     

A modified method of UDS detection in vitro suitable for screening the DNA-damaging effects of chemicals

The UDS induced in cultured FL cells by exposure to chemicals was measured as hydroxyurea-resistant incorporation of 3H-TdR in the acid-insoluble fraction of the 14C-TdR-prelabelled cells synchronized by the combination of arginine starvation and pretreatment with hydroxyurea. The level of UDS is represented by the ratios of 3H/14C radioactivities which are measures of specific activities of 3H. Two direct-acting alkylating agents, MMS and MNNG, a cross-linking agent, mitomycin C, and 3 procarcinogens, B(a)P, AFB1 and cyclophosphamide elicited UDS in the absence or presence of the liver-metabolizing system. Three chemicals of unknown carcinogenicity were also able to induce UDS in this assay system, i.e., bis-(O,O-diethylphosphinothioyl)-disulphide, 4-chlorophenoxy acetic acid (sodium salt) and caramelized malt sugar. With the exception of 4-chlorophenoxy acetic acid, they were also active in the Ames test.     

Preparation of [B23-D-alanine]des-(B25-B30)-hexapeptide-insulin by a combination of enzymic and non-enzymic synthesis.

Des-(B25-B30)-hexapeptide-insulin with B23-glycine replaced by D-alanine was prepared by a combination of enzymic and non-enzymic syntheses. The purified product was homogeneous in polyacrylamide-gel electrophoresis and could be crystallized. The biological activity in vivo of crystalline [B23-D-Ala]des-(B25-B30)-hexapeptide-insulin was determined as 58% of that of standard pig insulin (27 i.u./mg).