Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.     

Two new flavone glycosides from Cirsium lineare

An examination of four species of Cirsium disclosed the presence of two new flavonoids in C. lineare. The structure of one was 5,4′-dihydroxy-6,7,3′-trimethoxyflavone (cirsilineol) 4′-monoglucoside and the other 5,3′,4′-trihydroxy-6,7-dimethoxyflavone (cirsiliol) 4′-monoglucoside. Luteolin 7-glucoside was found in C. suffultum, and pectolinarin and linarin in C. kamtschaticum and C. pectinellum.     

Reduced Visual Cortex Gray Matter Volume and Thickness in Young Adults Who Witnessed Domestic Violence during Childhood

Exposure to interparental violence is associated with negative outcomes, such as depression, post-traumatic stress disorder and reduced cognitive abilities. However, little is known about the potential effects of witnessing domestic violence during childhood on gray matter volume (GMV) or cortical thickness. High-resolution 3.0 T volumetric scans (Siemens Trio Scanner) were obtained on 52 subjects (18–25 years) including 22 (6 males/16 females) with a history of visually witnessing episodes of domestic violence, and 30 (8 males/22 females) unexposed control subjects, with neither a current nor past DSM-IV Axis I or II disorder. Potential confounding effects of age, gender, level of parental verbal aggression, parental education, financial stress, full scale IQ, and total GMV, or average thickness were modeled using voxel based morphometry and FreeSurfer. Witnessing domestic violence subjects had a 6.1% GMV reduction in the right lingual gyrus (BA18) (P = 0.029, False Discovery Rate corrected peak level). Thickness in this region was also reduced, as was thickness in V2 bilaterally and left occipital pole. Theses regions were maximally sensitive to exposure to witnessing domestic violence between 11–13 years of age. Regional reductions in GMV and thickness were observed in both susceptible and resilient witnessing domestic violence subjects. Results in subjects witnessing domestic violence were similar to previously reported results in subjects with childhood sexual abuse, as the primary region affected was visual cortex. Brain regions that process and convey the adverse sensory input of the abuse may be specifically modified by this experience, particularly in subjects exposed to a single type of maltreatment. Exposure to multiple types of maltreatment is more commonly associated with morphological alterations in corticolimbic regions. These findings fit with preclinical studies showing that visual cortex is a highly plastic structure.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

Primary structure of an N-linked sugar chain derived from glucoamylase of Rhizopus niveus

The primary structure of the N-linked sugar chain of Rhizopus niveus glucoamylase (major component) was investigated. The carbohydrate moiety was released from the polypeptide backbone by Flavobacterium sp. endo-beta-N-acetylglucosaminidase digestion. Studies using the method of exoglycosidase digestion of the fluorescent pyridylamino derivative, gel-permeation chromatography on Bio-Gel P-4 and 400-MHz 1H-NMR spectroscopy revealed that the most abundant structure is (Man)8-GlcNac-ol.     

A complete deletion mutant of the Escherichia coli dnaKdnaJ operon

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.