Amino acid sequence homology in 30 S ribosomal protein S19 from Bacillus stearothermophilus and Escherichia coli.

The amino therminal sequence of 30 S ribosomal protein S19 from Bacillus stearothermophilus has been determined. Comparison with the corresponding ribosomal protein from Escherichia coli reveals substantial sequence homology in the first thirty residues and provides further evidence for conservation of genetic information which specifies the primary structure of ribosomal proteins in these taxonomically dissimilar organisms.

Exercise-stimulated glucose turnover in the rat is impaired by glucosamine infusion.

The infusion of glucosamine causes insulin resistance, presumably by entering the hexosamine biosynthetic pathway; it has been proposed that this pathway plays a role in hyperglycemia-induced insulin resistance. This study was undertaken to determine if glucosamine infusion could influence exercise-stimulated glucose uptake. Male SD rats were infused with glucosamine at 0.1 mg x kg(-1) x min(-1) (low-GlcN group), 6.5 mg x kg(-1) x min(-1) (high-GlcN group), or saline (control group) for 6.5 h and exercised on a treadmill for 30 min (17 m/min) at the end of the infusion period. Glucosamine infusion caused a modest increase in basal glycemia in both experimental groups, with no change in tracer-determined basal glucose turnover. During exercise, glucose turnover increased approximately 2.2-fold from 46 +/- 2 to 101 +/- 5 pmol x kg(-1) x min(-1) in the control group. Glucose turnover increased to a lesser extent in the glucosamine groups and was limited to 88% of control in the low-GlcN group (47 +/- 2 to 90 +/- 3 pmol x kg(-1) x min(-1); P < 0.01) and 72% of control in the high-GlcN group (43 +/- 1 to 73 +/- 3 pmol kg(-1) 1 min(-1); P < 0.01). Similarly, the metabolic clearance rate (MCR) in the control group increased 72% from 6.1 +/- 0.2 to 10.5 +/- 0.7 ml kg(-1) x min(-1) in response to exercise. However, the increase in MCR was only 83% of control in the low-GlcN group (5.2 +/- 0.5 to 8.7 +/- 0.5 ml x kg(-1) x min(-1); P < 0.01) and 59% of control in the high-GlcN group (4.5 +/- 0.2 to 6.2 +/- 0.3 ml x kg(-1) x min(-1); P < 0.01). Neither glucosamine infusion nor exercise significantly affected plasma insulin or free fatty acid (FFA) concentrations. In conclusion, the infusion of glucosamine, which is known to cause insulin resistance, also impaired exercise-induced glucose uptake. This inhibition was independent of hyperglycemia and FFA levels.

TNF-alpha-induced insulin resistance in vivo and its prevention by troglitazone.

Tumor necrosis factor (TNF)-alpha may play a role in the insulin resistance of obesity and NIDDM. Troglitazone is a new orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsulinemia in both diabetic animal models and NIDDM subjects. To determine whether this drug could prevent the development of TNF-alpha-induced insulin resistance, glucose turnover was assessed in rats infused with cytokine and pretreated with troglitazone. Normal male Sprague-Dawley rats were fed normal powdered food with or without troglitazone as a food admixture (0.2%). After approximately 10 days, rats were infused with TNF-alpha for 4-5 days, producing a plasma concentration of 632 +/- 30 pg/ml. In vivo insulin action was measured by the euglycemic-hyperinsulinemic clamp technique at a submaximal (24 micromol x kg[-1] x min[-1]) and maximal insulin infusion rate (240 micromol x kg[-1] x min[-1]). TNF-alpha infusion resulted in a pronounced reduction in submaximal insulin-stimulated glucose disposal rate (GDR) (97 +/- 10 vs. 141 +/- 4 micromol x kg[-1] x min[-1], P < 0.05), maximal GDR (175 +/- 8 vs. 267 +/- 6 micromol x kg[-1] x min[-1], P < 0.01), and in insulin receptor-tyrosine kinase activity (IR-TKA) (248 +/- 39 vs. 406 +/- 32 fmol ATP/fmol IR, P < 0.05). It also led to a marked increase in basal insulin (90 +/- 24 vs. 48 +/- 6 micromol/l, P < 0.05) and free fatty acid (FFA) concentration (2.56 +/- 0.76 vs. 0.87 +/- 0.13 mmol/l, P < 0.01). Troglitazone treatment completely prevented the TNF-alpha-induced decline in submaximal GDR (133 +/- 16 vs. 141 +/- 4 micromol x kg[-1] x min[-1], NS) and maximal GDR (271 +/- 19 vs. 267 +/- 6 micromol x kg[-1] x min[-1], NS). The hyperlipidemia was partially corrected by troglitazone (1.53 +/- 0.28 vs. 0.87 +/- 0.13 mmol/l, P < 0.05), while IR-TKA and insulin concentration remained unaffected by the drug. Troglitazone restores insulin action possibly by lowering the FFA concentration of the blood and/or by stimulating glucose uptake at an intracellular point distal to insulin receptor autophosphorylation in muscle. If TNF-alpha plays a role in the development of the obesity/NIDDM syndrome, troglitazone may prove useful in its treatment.

Troglitazone prevents hyperglycemia-induced but not glucosamine-induced insulin resistance.

Hyperglycemia can lead directly to a secondary state of insulin resistance or can worsen a preexisting insulin-resistant state. Troglitazone is an orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsulinemia in both diabetic animal models and NIDDM subjects. To determine whether this drug could prevent the development of hyperglycemia-induced insulin resistance and to investigate the mechanism by which this might occur, we studied troglitazone's effect on insulin action in rats made hyperglycemic or infused with glucosamine. Normal male SD rats were fed regular powdered diet with or without troglitazone as a food admixture (0.2%). After 2 weeks, rats were made hyperglycemic with glucose (52 mg x kg(-1) x min[-1]) and somatostatin (0.8 microg x kg(-1) x min[-1]) infusion or were infused with glucosamine (6.5 mg x kg(-1) x min[-1]) for 6.5 h. In vivo insulin action was measured by the hyperinsulinemic-euglycemic clamp technique at a submaximal (24 pmol x kg(-1) x min[-1]) or maximal (240 pmol x kg(-1) x min[-1]) insulin infusion rate. The infusion of glucose and somatostatin caused a pronounced rise in the plasma glucose concentration (19.8 +/- 0.6 mmol/l) compared with saline-infused animals (8.0 +/- 0.2 mmol/l; P < 0.001). Hyperglycemia resulted in insulin resistance, as evidenced by a marked reduction in the submaximal glucose disposal rate (GDR) (78 +/- 7 vs. 135 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) and maximal GDR (141 +/- 9 vs. 237 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) compared with the control group. Troglitazone treatment largely prevented the hyperglycemia-induced decline in submaximal (116 +/- 7 micromol x kg(-1) x min[-1]) and maximal GDR (209 +/- 9 micromol x kg(-1) x min(-1); P < 0.05). Glucosamine infusion also resulted in a marked reduction in the submaximal GDR (85 +/- 3 vs. 135 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) and maximal GDR (137 +/- 14 vs. 237 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) compared with the control group. In contrast to the results in the hyperglycemic animals, troglitazone treatment had no effect on glucosamine-induced insulin resistance. In summary, 1) in normal rats, experimental hyperglycemia, as well as glucosamine infusion, led to a marked state of peripheral and hepatic insulin resistance; 2) troglitazone treatment prevented the hyperglycemia-induced, but not the glucosamine-induced, insulin resistance; and 3) either troglitazone acts at one or more sites proximal to the entry of glucosamine into the hexosamine pathway, or the increased flux of glucose-derived products through the hexosamine pathway is not a major mechanism for the hyperglycemia-induced defect in insulin action in these animals.

Possible origin of murine AIDS-inducing sequence.

The murine AIDS (MAIDS) virus has a unique sequence in its gag p12 region. A transcript which hybridizes with this sequence is expressed in normal C57BL/6 mice. This transcript has been proposed to be the origin of the MAIDS virus, since the virus was originally isolated from radiation-induced leukemic C57BL/6 mice. The transcript, designated Edv, was molecularly cloned and sequenced. Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic defective MAIDS virus has 16-bp deletions and a 1-bp insertion in the 5' and 3' regions of the gag p12 sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the gag p12 region of the MAIDS virus is less homologous to that of the helper virus and Edv transcript due to the frameshift mutations. This suggested that the MAIDS virus was generated by such frameshift mutations in the gag p12 region during recombination between the helper virus and the Edv or a related sequence.

Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens.

1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.

Generation of a polymorphic marker linked to thymoma susceptibility gene of rat 1 by genetically-directed representational difference analysis.

BUF/Mna (BUF) is a rat strain susceptible to spontaneous development of thymomas. We have previously shown that the thymoma susceptibility is controlled principally by a dominant susceptibility gene located on chromosome 7, thymoma susceptibility gene of rat 1 (Tsr1). To generate genetic markers tightly linked to Tsr1, we performed genetically directed representational difference analysis (GDRDA) with three combinations of the tester and driver DNAs. From 124 ¿ACI/NMs x (BUF x ACI/NMs) F1¿ backcross rats, 12 rats with the ACI/BUF genotype in the Tsr1 region (A/B rats) and 13 rats with the ACI/ACI genotype in the region (A/A rats) were selected, and their DNAs were pooled, respectively. Three kinds of tester DNAs, i) inbred BUF, ii) (BUF x ACI)F1, and iii) the pool from the A/B rats, were subtracted by the driver DNA prepared from the pool of the A/A rats. The three combinations yielded one, two, and one polymorphic marker(s), respectively. One marker, D7Ncc28, was isolated commonly by the three combinations of subtraction, and another marker, D11Ncc12 was isolated only by the second combination. Linkage analysis demonstrated that D7Ncc28 was located in the 8.3 cM region where Tsr1 has been mapped. The three combinations of subtraction were shown to be almost equally capable of isolating polymorphic markers in a specific chromosomal region.

Isolation and characterization of fourteen ribosomal proteins from small subunits of yeast.

A method for preparation of a large amount of ribosomal subunits from Saccharomyces cerevisiae by a Ti-15 zonal rotor is described. The proteins of the small subunits (ca. 50 000 A260 units) were separated into 22 fractions by chromatography on carboxymethylcellulose columns. Fourteen proteins were then purified from the ten chromatographic fractions by filtration through Sephadex G-100 or Sephacryl S--200. The isolated proteins are YP 6, YP 7, YP 9, YP 12, YP 14', YP 14'', YP 28, YP 38, YP 45, YP 50, YP 52, YP 58, YP 63, and YP 70. The molecular weight and amino acid compositions of these proteins are presented.

Tourist C transposable elements are closely associated with genes expressed in flowers of rice (Oryza sativa).

Tourist elements comprise a group of transposable elements in plants. One of these elements, Tourist-OsaCatA(a Tourist C element), has been found in the 5; flanking region of a catalase gene, CatA, in rice (Oryza sativa). Using reverse transcriptase-PCR (RT-PCR) analyses of leaves, roots, flowers and developing seeds of rice, we assessed the transcription levels of ten known genes containing Tourist C elements, and of three additional putative genes for which expressed sequence tags (ESTs) including Tourist C elements have been isolated. We found that nine of the ten known genes and two of the three represented by ESTs were expressed in at least one of the organs we analyzed, and all of the genes detected were expressed in flowers, usually in stamens or pistils. We also assessed the expression of the 29 Tourist C-containing hypothetical coding sequences (CDSs) obtained so far by high-throughput genomic sequencing. We found that CDSs of all 11 genes whose transcripts were detectable by RT-PCR were expressed in flowers, especially in stamens or pistils. In contrast, RT-PCR analyses of genes or CDSs associated with other miniature inverted-repeat transposable elements (MITEs), such as Tourist D, Gaijin, Explorer, and Castaway, showed that some of them were expressed only minimally or not at all in flowers. Therefore, compared with other MITEs, Tourist C elements seem to show a strong association with genes that are expressed in the flowers of rice.

The novel thromboxane A2 receptor antagonist KW-3635 abolishes the cyclic flow reduction in the canine carotid artery.

We tested the hypothesis that the abolition of the cyclic flow reduction (CFR) in the canine carotid artery is related to inhibition of ex vivo platelet aggregation following administration of KW-3635, a thromboxane A2 receptor antagonist, or aspirin. The CFR was induced in the carotid artery of anesthetized dogs by mechanical injury and narrowing of the artery. After induction of CFR, KW-3635 or aspirin was administered every 30 min at doses of 0,1,0.3, 1 and 3 mg/kg (i.v.). The ex vivo platelet aggregation, induced by sodium arachidonate and collagen, was also examined before and 15 min after each administration. KW-3635 and aspirin, at doses of 1 mg/kg i.v. and above, inhibited CFR and ex vivo platelet aggregation. These results that CFR in the canine carotid artery is platelet-dependent.

Inhibitory effect of KW-3635, a new thromboxane A2-receptor antagonist, on arterial thrombosis in guinea pigs.

The antithrombotic effects of the thromboxane (TX) A2-receptor antagonist and aspirin were determined using a photochemically-induced arterial thrombosis model in the femoral arteries of guinea pigs. KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]-oxepine-2-carboxylate monohydrate) and BM-13505, both of which are TXA2-receptor antagonists, and aspirin inhibited the thrombus formation at the doses that inhibited the ex vivo platelet aggregation induced by sodium arachidonate (100 microM) or collagen (3 micrograms/ml). These results support the notion that TXA2 is an important mediator in the present model of arterial thrombogenesis.

Effects of a thromboxane A2-receptor antagonist, a thromboxane synthetase inhibitor and aspirin on prostaglandin I2 production in endothelium-intact and -injured aorta of guinea pigs.

We examined the effects of KW-3635, a thromboxane (TX) A2-receptor antagonist, and OKY-046, a TX synthetase inhibitor, on the prostaglandin (PG) I2 production in endothelium-intact and -injured guinea pig aorta and compared them with those of aspirin. In the endothelium-intact aorta, both the low (3 mg/kg) and the high (100 mg/kg) dose of aspirin similarly reduced the PGI2 production, as measured ex vivo 1 hr after the injury. In contrast, neither KW-3635 (10 mg/kg) nor OKY-046 (30 mg/kg) inhibited the PGI2 production. The endothelial injury, induced by balloon catheterization, caused a reduction of PGI2 production in the aorta and decline of plasma PGI2/TXA2 ratio. In the endothelium-injured animals, the high dose of aspirin further reduced the PGI2 production in the aorta, whereas KW-3635 and OKY-046 did not affect it. KW-3635 and OKY-046 also ameliorated the reduced ratio of PGI2/TXA2 in the plasma. The present results demonstrate that aspirin, but not KW-3635 or OKY-046, reduces the PGI2 production in the aorta either in the endothelium-intact or -injured state. It is thus suggested that the TXA2-receptor antagonist and the TX synthetase inhibitor have some advantages over aspirin when used for the prevention of acute thrombosis after percutaneous transluminal angioplasty.

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene.

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5'-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5'-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects. The 1.9 kb 5'-upstream fragment (-1559 to +342) of the CatA gene was fused with the Escherichia coli beta-glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between -1564 to -699. Abscisic acid (ABA) at a final concentration of 10(-6) M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5'-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at -266 to -254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light. The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5alpha, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.

Changes in translatable mRNA populations induced in rice seedlings by exposure to freeze-thaw stress.

In order to examine whether changes in gene expression are induced by injury in seedlings of rice (Oryza sativa) after a lethal freeze-thaw stress. mRNAs were extracted and their in vitro translation products were compared with those of the control plants by polyacrylamide gel electrophoresis. An mRNA encoding an entirely new protein with M(r) of ca. 37 kDa was detected as early as 30 min of thawing at 28 degrees C. Two-dimensional gel electrophoresis revealed the induction of mRNAs for at least 10 additional proteins, including those in a range of 15 to 20 kDa. Most of these proteins appear to be distinct from heat-shock proteins. The steady state level of mRNAs encoding putative major light-harvesting chlorophyll a/b binding protein-I and -II were, on the other hand, very low as compared with those of the control plants. The changes observed resemble those previously seen in cold-sensitive plants exposed to sub-lethal chilling temperatures (4-10 degrees C).

Characteristics of [3H]nimodipine binding to sarcolemmal membranes from rat vas deferens and its regulation by guanine nucleotide.

The binding properties of a 1,4-dihydropyridine (DHP) calcium entry blocker, [3H]nimodipine, to a microsomal fraction from rat vas deferens was characterized. The specific binding was saturable, rapid and reversible. Scatchard analysis of the binding revealed a single binding site, and the dissociation constant and the maximum number of binding sites were 0.31 +/- 0.02 nM and 97.0 +/- 7.19 fmol/mg protein, respectively. Both the Kd value obtained from the kinetic study and the IC50 value from relaxation of the K+-depolarized organ were approximately 0.4 nM, indicating that the binding site is closely related to the functional Ca2+ channel. The specific [3H]nimodipine binding was displaced by DHP derivatives at low concentration and by verapamil at high concentration, but diltiazem had no effect on the binding. Calcium chelating agents decreased the [3H]nimodipine binding which was restored by adding Ca2+. 5'-Guanylylimidodiphosphate caused a rightward shift of the displacement curve for Bay K 8644 but not for nimodipine, suggesting the involvement of guanine nucleotide binding protein in the signal transduction between the DHP binding site and the Ca2+ channel.

Yeast ribosomal proteins. VIII. Isolation of two proteins and sequence characterization of twenty-four proteins from cytoplasmic ribosomes.

Two proteins, YL41 and YL43, were isolated from 80S ribosomes of Saccharomyces cerevisiae by filtration through a Sephacryl S-200 column and by chromatography on a column of carboxymethylcellulose. Their amino acid compositions are presented. Twenty-four proteins including these two proteins were subjected to sequence analyses by automated Edman degradation. Amino-terminal amino acid sequences were determined for 17 proteins,YS3, YS9, YS23, YS24, YS29, YL6, YL8, YLll, YLI5,YL17, YL23, YL28, YL33, YL37, YL39, YL41, and YL43.YL41, which has a 72.7% lysine and arginine content, was found to be particular to eukaryotic ribosomes. The amino-termini of another seven proteins, YS2, YS5, YS8, YS12,YS13, YS20, and YS27, were suggested to be blocked. Comparison of the amino-terminal sequences with all other ribosomal protein sequences so far available indicates that YS9 shows sequence homology to rat liver ribosomal protein S8 (Wittmann-Liebold et al. 1979).

Isolation and characterization of twenty-three ribosomal proteins from large subunits of yeast.

The proteins of large ribosomal subunits from Saccharomyces cerevisiae were separated into 25 fractions by chromatography on columns of carboxymethylcellulose (CMC). Twenty-three proteins were then purified from the 12 CMC fractions by filtration through Sephadex G-75, Sephadex G-100, and Sephacryl S-200, and/or by phosphocellulose column chromatography. The isolated proteins are YP 1, YP 2, YP 9, YP 11, YP 13', YP 16, YP 18, YP 26, YP 39, YP 41, YP 42, YP 42', YP 44, YP 45, YP 47', YP 52a, YP 53, YP 55, YP 59, YP 62, YP 68, YP A1, and YP A2. The molecular weight and amino acid composition of these proteins are presented.