Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.     

Rapid isolation of lysyl-tRNA synthetase from rat liver

The high molecular weight aminoacyl-tRNA synthetase complex (the 24S complex) was isolated from rat liver by ultracentrifugation. The lysyl-tRNA synthetase (E.C. 6.1.1.6) was selectively dissociated by hydrophobic interaction chromatography on 1,6 diaminohexyl agarose followed by hydroxylapatite chromatography and DEAE chromatography. The lysyl-tRNA synthetase dissociated from the 24S synthetase complex was purified approximately to 2700 fold with 14% yield.     

Crystallization and preliminary data of Indian buffalo erythrocyte carbonic anhydrase

The most abundant anhydrase isoenzyme from the erythrocyte of Indian buffalo has been purified using affinity gel and DEAE-cellulose ion-exchange columns and single crystals suitable for X-ray diffraction studies have been obtained. The unit cell dimensions are a = 46.8 A, b = 104.5 A, c = 60.4 A, beta = 91.2 degrees and the space group is P2(1), with two molecules per asymmetric unit.     

Enhancement of water status by calcium pretreatment in groundnut and cowpea plants subjected to moisture stress

Summary The effect of calcium in the water relations and tolerance to moisture deficits was tested in groundnut and cowpea. In both species, enrichment of tissue with calcium resulted in maintenance of a higher water status under stress associated with low proline accumulation. The extent of membrane damage (as reflected by the absorbance at 273 nm) was lesser in leaves of plants fed with higher levels of Ca⁺⁺ when subjected to simulated stress. The rate of water loss from the leaves of Ca⁺⁺-enriched plants was also lower. The possible role of Ca⁺⁺ in inducing membrane stability and maintenance of higher water status is discussed.     

Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver

The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction.     

Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.     

Synthesis and biologic activity of 3 beta-thiovitamin D3

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.     

A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants

A simple method was developed for the controlled cleavage of protein disulfide bonds and the simultaneous blockage of the free sulfhydryl groups in the absence of a denaturant. The disulfide bonds of bovine serum albumin were cleaved unsymmetrically at pH 7.0 using 0.1 M sulfite in 0.1 M phosphate buffer and the free sulfhydryl groups formed were sulfonated in an oxidation-reduction cycle using molecular oxygen and 400 microM cupric sulfate as a catalyst. The reaction was affected by cupric ion concentration, sulfite concentration, reaction pH and temperature. The standardized method was successfully used to cleave the disulfide bonds of other proteins pepsin, trypsin, and chymotrypsin. The method is reliable and can be used for achieving progressive cleavage of disulfide bonds in proteins without employing a denaturant.