Study of the maturation of 5 s RNA precursors in Escherichia coli

The existence of three precursors to 5 s RNA's (p5 s RNA's) has been confirmed. p5 s RNA's and mature 5 s RNA have different 5′-terminal sequences and produce the following 5′-terminal oligonucleotides: p5 s RNA-I:pAUUUG; p5 s RNA-II:pUUUG; p5 s RNA-III:pUUG; 5 s RNA:pUG. The results of experiments on pulse-labelled cells treated with actinomycin D, on chloramphenicol-inhibited cells, on various ribosome assembly-defective mutants and on the state of 5 s RNA in polysomes after a short labelling period, support the following conclusions. (1) p5 s RNA-I is the first identifiable precursor which appears during a pulse. (2) The amount of p5 s RNA-II, relative to those of the other p5 s RNA's, is very low at all times during pulse-chase experiments. On the contrary, it becomes significant during chloramphenicol inhibition and in one assembly-defective mutant under non-permissive conditions. (3) The maturation steps which lead to p5 s RNA-III and 5 s RNA normally occur after binding to 50 s subunit precursor particles and are, consequently, dependent upon protein synthesis. (4) The transition from p5 s RNA-III to 5 s RNA is a slow process, which is neither dependent upon nor required for the proper functioning of 50 s subunits in protein synthesis.     

Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s)

To analyze functional differences in the interactions of the glucagon receptor (GR) with the two predominant splice variants of Galpha(s), GR was covalently linked to the short and the long forms Galpha(s)-S and Galpha(s)-L to produce the fusion proteins GR-Galpha(s)-S and GR-Galpha(s)-L. GR-Galpha(s)-S bound glucagon with an affinity similar to that of GR, while GR-Galpha(s)-L showed a 10-fold higher affinity for glucagon. In the presence of GTPgammaS, GR-Galpha(s)-L reverted to the low affinity glucagon binding conformation. Both GR-Galpha(s)-L and GR-Galpha(s)-S were constitutively active, causing elevated basal levels of cAMP even in the absence of glucagon. A mutant GR that failed to activate G(s) (G23D1R) was fused to Galpha(s)-L. G23D1R-Galpha(s)-L bound glucagon with high affinity, but failed to elevate cAMP levels, suggesting that the mechanisms of GR-mediated Galpha(s)-L activation and Galpha(s)-L-induced high affinity glucagon binding are independent. Both GR-Galpha(s)-S and GR-Galpha(s)-L bound the antagonist desHis(1)[Nle(9),Ala(11),Ala(16)]glucagon amide with affinities similar to GR. The antagonist displayed partial agonist activity with GR-Galpha(s)-L, but not with GR-Galpha(s)-S. Therefore, the partial agonist activity of the antagonist observed in intact cells appears to be due to GRs coupled to Galpha(s)-L. We conclude that Galpha(s)-S and Galpha(s)-L interact differently with GR and that specific coupling of GR to Galpha(s)-L may account for GTP-sensitive high affinity glucagon binding.     

Functional analysis of activated C1s, a subcomponent of the first component of human complement, by monoclonal antibodies

Three mouse monoclonal antibodies (M365, M81, and M241) directed against human C1s were used to analyze the structure of C1s related to the enzymatic activity. M365 and M81 recognized different epitopes on the heavy chain of C1s and could bind to C1s, as well as to C1s. The C4 cleaving activity of C1s was completely blocked by M81 and was partially blocked by M365. Although the C2 cleaving activity of C1s was partially inhibited by M81, no blocking was observed with M365. Both antibodies had no effect on the esterolytic activity of C1s. These results indicate that the C4 and C2 binding sites on C1s reside in the heavy chain, and they are distinct from each other. M241 could bind only to C1s, an active form of C1s. After reduction of C1s, M241 could not react with either heavy or light chain of C1s. The esterolytic activity of C1s was markedly reduced by M241. Furthermore, M241 blocked not only the cleavage of C4 and C2 by C1s but also the complex formation of C1s and C1 inactivator. From these observations, we suggest that M241 reacts with the active site of C1s, and both heavy and light chains of C1s participate in the composition of the active site.     

Spread of Heterobasidion annosum s.s. and Heterobasidion parviporum in Picea abies 15 years after stump inoculation

The tree pathogenic fungi Heterobasidion annosum s.s. and Heterobasidion parviporum cause root and butt rot in Norway spruce (Picea abies) and produce serious economic losses to the forest sector in Europe. We experimentally studied inter- and intraspecific differences between H. parviporum and H. annosum s.s. in the way they infect stumps and spread into neighbouring trees. Eleven H. parviporum and nine H. annosum s.s. isolates were artificially inoculated on stumps of two spruce stands after first thinning. After 15 years, the same isolates were reisolated from neighbouring trees. Heterobasidion parviporum spread more frequently from the inoculated stumps to the neighbouring trees than H. annosum s.s. The surroundings of H. annosum s.s. stumps that did not spread were often colonized by H. parviporum. Heterobasidion annosum s.s. spread was restricted mainly to the areas of the plot where no other Heterobasidion genotypes had been inoculated. In such cases, H. annosum s.s. tended to develop into bigger genets than H. parviporum. The probability of stump-to-tree spread of H. parviporum depended on the diameter of the stumps, suggesting that H. parviporum spread may relate to the presence of heartwood. Both H. parviporum and H. annosum s.s. proved to be strong pathogens on Norway spruce; however, when competing for the same trees, H. parviporum seemed capable of excluding H. annosum s.s. from the stand.     

Different Classes of Ribonucleic Acid Isolated from Lymphocytic Choriomeningitis Virus

Purified preparations of lymphocytic choriomeningitis virus (LCM virus) contain three classes of RNA. The previously described 18s, 23s, 28s, and 31s RNAs, where the 23s and 31s RNAs are viral-specific, and the 18s and 28s RNAs probably are host RNAs incorporated in the virion. Now, 4s, 5s, and 5.5s RNAs can be isolated as well. Thus five RNAs which migrate by acrylamide gel electrophoresis as ribosome-derived RNA can be isolated from purified LCM virus. This observation further supports the reports that arenaviruses may contain ribosomes. The ribosome-derived RNA can be synthesized both before and after the virus infection. The viral 23s could be a hydrogen-bonded complex forming the 31s RNA, or it could be contained in defective interfering LCM virus particles; these possibilities are examined.     

不同种源羊草的SOD、POD的活性及丙二醛含量的比较

 本文将内蒙古不同种源的羊草(Leymus chinensis)进行PEG水分胁迫处理后,分别进行POD和SOD的酶活性及MDA含量的测定,并算出各浓度梯度和时间梯度下的增量。并把POD、SOD及MDA的测试结果及其浓度梯度和时间梯度下的增量用NTS软件进行聚类分析,发现不同种源羊草的SOD、POD对PEG水分胁迫在时间和浓度上的反应是不同的,且植物体内MDA的积累量也是不同的,聚类分析进一步表明不同地理种群羊草按POD和SOD的酶活及MDA含量都可分成3大类,且所得结果相同,这与它们对各自自然生境的适应也是一致的。     

Replacement of the alpha5 helix of Galpha16 with Galphas-specific sequences enhances promiscuity of Galpha16 toward Gs-coupled receptors

G(16) can couple indiscriminately to a large number of G protein-coupled receptors (GPCRs), making it a prime candidate as a universal adaptor for GPCRs. In order to increase the promiscuity of Galpha(16), three chimeras incorporating increasing lengths of G(s)-specific residues (25, 44 or 81 residues) into the C-terminus of Galpha(16) were constructed and named 16s25, 16s44 and 16s81, respectively. The chimeras were examined for their ability to mediate receptor-induced stimulation of phospholipase C (PLC) and Ca(2+) mobilization. 16s25 was more effective than 16s44 and 16s81 at coupling to G(s)-linked receptors. 16s25 coupled productively to 10 different G(s)-coupled receptors examined and, for 50% of these receptors, 16s25-mediated PLC activities were higher than those mediated via Galpha(16). Similar results were observed for agonist-induced Ca(2+) mobilizations. These results show that incorporating the alpha5 helix of Galpha(s) into Galpha(16) can increase the promiscuity of 16s25 towards G(s)-coupled receptors.     

Is carbon dioxide (CO2) a useful short acting anaesthetic for small laboratory animals?

The anaesthetic effect of carbon dioxide (CO2) was investigated under predetermined exposure times in rats, mice and guinea pigs with admixture of 20% of oxygen (O2), and with 20% of ambient air in rats. In rats first symptoms (median) were detectable between 7 and 9.5 s, the induction time (median) varied between 16 and 20.5 s and the surgical tolerance (median) was 40 s (after 60 s of exposure) and 53.5 s (after 120 s of exposure) to 80% CO2/20% O2. When O2 was replaced by ambient air, a surgical tolerance of 53.5 s (after 60 s of exposure) and 77 s (after 120 s of exposure) was measured. In mice the induction time to 80% CO2/20% O2 was 10 s and the surgical tolerance 19.5 s (after 120 s of exposure). Guinea pigs showed an induction period of 20 s and a surgical tolerance of 50 s (after 30 s of exposure) to 80% CO2/O2. Recovery was short and smooth in all species. This method of general anaesthesia seems to be suitable for short and painful interventions, mainly in rats, but also in guinea pigs.     

河南省淮河流域鳑鲏亚科5种鱼形态差异分析

为进一步从可量性状比较中华鳑鲏(Rhodeus sinensis)、越南鱊(Acheilongnathus tonkinensis)、大鳍鱊(A. macropterus)、兴凯鱊(A. chankaensis)和斑条鱊(A. taenianalis)的种间形态差异, 丰富鳑鲏亚科鱼类的形态分类特征, 研究采用多变量形态度量学方法对河南省淮河流域这5种鱼的形态进行比较分析。结果表明: 这5种鱼在体宽/体长、头长/体长、吻长/体长、尾柄长/体长、背鳍基底长/体长、腹鳍长/体长、臀鳍长/体长和头长/吻长8个比值性状上存在极显著性差异(P< 0.01), 在全长/体长、体高/体长2个比值性状上存在显著性差异(P<0.05), 这些差异主要集中在鱼体的头部、尾部和鳍等部位。主成分分析、聚类分析和判别分析显示5种鱼存在显著性形态差异, 斑条鱊与越南鱊的差异程度最大, 与大鳍鱊差异程度最小。判别分析获得的判别准确率在82.35%—100.00%变化, 可以从一定的角度区分这5种鱼。     

The phosphate groups of the high mobility group like protein P1 strengthens its affinity for DNA.

PCA soluble proteins isolated from rat liver and proliferating HeLa interphase cells were subjected to chromatography on columns containing immobilized s.s and d.s. DNA. P1 from rat liver was eluted from s.s. and d.s. DNA between 0.20 and 0.45 M NaCl, while dephosphorylated P1 was not retained by s.s. and d.s. DNA columns at 0.25 M, suggesting that phosphate groups enhance the affinity of P1 for DNA. P1 from proliferating HeLa interphase cells exhibit increased affinity for d.s. as well as s.s. DNA when compared to rat liver P1. The higher extent of phosphorylation in proliferating cells supports the finding that phosphate enhances rather than reduces the affinity of P1 for DNA.     

音频刺激与光耦合激发蝗虫趋光响应的效应

目的：研究光声耦合和对照光激发蝗虫趋光响应试验,为蝗虫的光电诱导捕集治理及趋光增益调控激发技术提供理论基础。方法：依据蝗虫趋光机理和声频刺激激发蝗虫的响应特性,利用LED光源、声频播放设备和蝗虫行为试验装置,进行了蝗虫对光声耦合和光谱光照趋光响应的对比测定,并探讨了光声耦合对蝗虫趋光效果影响的机理。结果：（1）蝗虫光声感受器对光能和声能接受和神经处理方式的不同,光谱光照和声频耦合刺激激发蝗虫生物活性和趋光v向应的双重叠加效应,增强了蝗虫的趋光活性,强化了蝗虫的趋光行为,提高了蝗虫的趋光响应,达到了推拉驱动蝗虫趋光响应的效应;（2）在光声耦合激发蝗虫趋光响应峰值上,蝗虫对不同声刺激的敏感性参数不同;（3）蝗虫对声刺激敏感参数接受的容限性,导致光谱光照在蝗虫诱导响应行为中起主导作用,而声刺激则起驱动激发蝗虫趋光响应的增益效应。结论：光谱光照和声刺激的合理布置和组合,能够有效提高蝗虫的趋光诱导响应效果。     

Expression of the mitochondrial genome in HeLa cells : I. Properties of the discrete RNA components from the mitochondrial fraction

The 16 s, 12 s and 4 s RNA components from the mitochondrial fraction of HeLa cells have been analyzed as to their sedimentation and electrophoretic properties, kinetics of labeling, metabolic stability, response to inhibitors of RNA synthesis, nucleotide composition and methylation level. After denaturation by heat-formaldehyde treatment, the 16 s and 12 s species sediment in sucrose gradients in the presence of formaldehyde as homogeneous components running behind 18 s RNA. Relative to the 28 s and 18 s RNA markers, the 16 s and 12 s RNA components behave in polyacrylamide gel electrophoresis as expected, in the absence of conformational influences, for a species slightly larger than 18 s RNA (i.e. with a molecular weight about 0.7 × 10⁶) and respectively, for a species with a molecular weight of about 0.4 × 10⁶. The 16 s and 12 s RNA correspond to the “21 s” and “12 s” electrophoretic components previously described in HeLa cells. However, in contrast to what has been reported for the latter components, the 16 s and 12 s RNA have been found to be methylated; furthermore, these species appear to have a considerably longer half-life than previously surmised on the basis of their behaviour in the presence of ethidium bromide and evidence is presented suggesting that they are synthesised in equimolar amounts.     

Roles for leptin receptor/STAT3-dependent and -independent signals in the regulation of glucose homeostasis

Leptin activates the long form of the leptin receptor (LRb) to control feeding and neuroendocrine function and thus regulate adiposity. While adiposity influences insulin sensitivity, leptin also regulates glucose homeostasis independently of energy balance. Disruption of the LRb/STAT3 signal in s/s mice results in hyperphagia, neuroendocrine dysfunction, and obesity similar to LRb null db/db mice. Insulin resistance and glucose intolerance are improved in s/s compared to db/db animals, however, suggesting that LRb/STAT3-independent signals may contribute to the regulation of glucose homeostasis by leptin. Indeed, caloric restriction normalized glycemic control in s/s animals, but db/db mice of similar weight and adiposity remained hyperglycemic. These differences in glucose homeostasis were not attributable to differences in insulin production between s/s and db/db animals but rather to decreased insulin resistance in s/s mice. Thus, in addition to LRb/STAT3-mediated adiposity signals, non-LRb/STAT3 leptin signals mediate an important adiposity-independent role in promoting glycemic control.     

Starved Tetrahymena Synthesize and Maintain an Excess of 5s rRNA in Their Cytoplasm1

We have measured the rate of accumulation of newly synthesized 5s ribosomal RNA (5s rRNA) in Tetrahymena thermophila cells in early log phase growth and in cells that had been starved in a dilute salt solution. From these measurements we have determined the rates of synthesis and levels of accumulation of 5s rRNA relative to 5.8s rRNA in these two different cell populations. In growing cells 5s rRNA is transcribed and accumulated in a 1:1 molar ratio when compared with 5.8s rRNA. In contrast, in starved cells, 5s rRNA is produced at a rate which is about 15% higher than that seen for 5.8s rRNA. This excess 5s rRNA accumulates in the cytoplasm in a non-ribosomal form and is maintained in the cell as long as the cell remains in a starved condition. The role this excess 5s rRNA may play in the control of 5s rRNA gene expression is discussed.     

Men report stronger attraction to femininity in women's faces when their testosterone levels are high

Many studies have shown that women's judgments of men's attractiveness are affected by changes in levels of sex hormones. However, no studies have tested for associations between changes in levels of sex hormones and men's judgments of women's attractiveness. To investigate this issue, we compared men's attractiveness judgments of feminized and masculinized women's and men's faces in test sessions where salivary testosterone was high and test sessions where salivary testosterone was relatively low. Men reported stronger attraction to femininity in women's faces in test sessions where salivary testosterone was high than in test sessions where salivary testosterone was low. This effect was found to be specific to judgments of opposite-sex faces. The strength of men's reported attraction to femininity in men's faces did not differ between high and low testosterone test sessions, suggesting that the effect of testosterone that we observed for judgments of women's faces was not due to a general response bias. Collectively, these findings suggest that changes in testosterone levels contribute to the strength of men's reported attraction to femininity in women's faces and complement previous findings showing that testosterone modulates men's interest in sexual stimuli.     

Synthesis and processing of high molecular weight RNA by nuclei isolated from embryos of Rana pipiens

When synthesized under conditions optimal for maximal methylation, two of the major classes of high molecular-weight RNA produced by isolated nuclei were indistinguishable from RNA purified from cytoplasmic ribosomes. The degree of methylation was found to influence the sedimentation velocity of only the heavier major RNA fraction. Upon becoming maximally methylated, this RNA sedimented at 27 s rather than 25 s. When comparing maximally-methylated nuclear RNA to cytoplasmic rRNA, it was found that on the basis of sedimentation coefficient, base composition and electrophoretic mobility, the 0.7 million dalton nuclear RNA was equivalent to 18 s cytoplasmic rRNA, the 1.5 million dalton nuclear RNA was equivalent to the 27 s moiety of 28 s cytoplasmic rRNA, and the 0.058 million dalton nuclear RNA was the same as the 7 s moiety of 28 s cytoplasmic rRNA. In isolated nuclei, the 27 s and 7 s RNA did not associate to form the 28 s duplex found in mature cytoplasmic ribosomes.     

Extra-long Gαs variant XLαs protein escapes activation-induced subcellular redistribution and is able to provide sustained signaling

Murine models indicate that Gαs and its extra-long variant XLαs, both of which are derived from GNAS, markedly differ regarding their cellular actions, but these differences are unknown. Here we investigated activation-induced trafficking of Gαs and XLαs, using immunofluorescence microscopy, cell fractionation, and total internal reflection fluorescence microscopy. In transfected cells, XLαs remained localized to the plasma membrane, whereas Gαs redistributed to the cytosol after activation by GTPase-inhibiting mutations, cholera toxin treatment, or G protein-coupled receptor agonists (isoproterenol or parathyroid hormone (PTH)(1-34)). Cholera toxin treatment or agonist (isoproterenol or pituitary adenylate cyclase activating peptide-27) stimulation of PC12 cells expressing Gαs and XLαs endogenously led to an increased abundance of Gαs, but not XLαs, in the soluble fraction. Mutational analyses revealed two conserved cysteines and the highly charged domain as being critically involved in the plasma membrane anchoring of XLαs. The cAMP response induced by M-PTH(1-14), a parathyroid hormone analog, terminated quickly in HEK293 cells stably expressing the type 1 PTH/PTH-related peptide receptor, whereas the response remained maximal for at least 6 min in cells that co-expressed the PTH receptor and XLαs. Although isoproterenol-induced cAMP response was not prolonged by XLαs expression, a GTPase-deficient XLαs mutant found in certain tumors and patients with fibrous dysplasia of bone and McCune-Albright syndrome generated more basal cAMP accumulation in HEK293 cells and caused more severe impairment of osteoblastic differentiation of MC3T3-E1 cells than the cognate Gαs mutant (gsp oncogene). Thus, activated XLαs and Gαs traffic differently, and this may form the basis for the differences in their cellular actions.     

Population structure of the Yersinia pseudotuberculosis complex according to multilocus sequence typing

Multilocus sequence analysis of 417 strains of Yersinia pseudotuberculosis revealed that it is a complex of four populations, three of which have been previously assigned species status [Y.?pseudotuberculosis sensu stricto (s.s.), Yersinia pestis and Yersinia similis] and a fourth population, which we refer to as the Korean group, which may be in the process of speciation. We detected clear signs of recombination within Y.?pseudotuberculosis s.s. as well as imports from Y.?similis and the Korean group. The sources of genetic diversification within Y.?pseudotuberculosis s.s. were approximately equally divided between recombination and mutation, whereas recombination has not yet been demonstrated in Y.?pestis, which is also much more genetically monomorphic than is Y.?pseudotuberculosis s.s. Most Y.?pseudotuberculosis s.s. belong to a diffuse group of sequence types lacking clear population structure, although this species contains a melibiose-negative clade that is present globally in domesticated animals. Yersinia similis corresponds to the previously identified Y.?pseudotuberculosis genetic type G4, which is probably not pathogenic because it lacks the virulence factors that are typical for Y.?pseudotuberculosis s.s. In contrast, Y.?pseudotuberculosis s.s., the Korean group and Y.?pestis can all cause disease in humans.     

Expression of the mitochondrial genome in HeLa cells : XIV. The relative positions of the 4 s RNA genes and of the ribosomal RNA genes in mitochondrial DNA

HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNA's have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.     

Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities

The tRNA(5-methylaminomethyl-2-thiouridine)-methyltransferase, which is involved in the biosynthesis of the modified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) present in the wobble position of some tRNAs, was purified close to homogeneity (95% purity). The molecular mass of the enzyme is 79,000 daltons. The enzyme activity has a pH optimum of 8.0-8.5, is inhibited by magnesium ions, and stimulated by ammonium ions. Two different intermediates in the biosynthesis of mnm5s2U34 are present in tRNA from the mutants trmC1 and trmC2. Unexpectedly, the product present in tRNA from trmC1 cells was identified by mass spectrometric and chromatographic analyses as 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), i.e. a more complex derivative than the final product mnm5s2U. The product present in tRNA from trmC2 cells was identified as 5-aminomethyl-2-thiouridine (nm5s2U). In the presence of S-adenosylmethionine the most purified enzyme fraction converts both cmnm5s2U34 and nm5s2U34 into mnm5s2U34. In the absence of S-adenosylmethionine, however, cmnm5s2U34 is converted into nm5s2U by this enzyme fraction. We conclude that the purified polypeptide has two enzymatic activities; one actually demodifies cmnm5s2U to nm5s2U and the other catalyzes the transfer of a methyl group from S-adenosylmethionine to nm5s2U, thus forming mnm5s2U. The sequential order of the biosynthesis of mnm5s2U34 is suggested to be: (Formula: see text). The molecular activity of the methyltransferase activity (nm5s2U34----mnm5s2U34) is 74 min-1, and the steady state concentration of the enzyme is only 78 molecules/genome equivalent in cells growing at a specific growth rate of 1.0/h.