Identification of nascent high density lipoproteins containing arginine-rich protein in human plasma.

A high density lipoprotein fraction accumulates in the plasma of patients with alcoholic hepatitis when a severe lecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for lecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism.     

Mice hypomorphic for Atr have increased DNA damage and abnormal checkpoint response

The ATR checkpoint pathway responds to DNA damage during the S/G2 phases of the cell cycle and is activated early in tumorigenesis. Investigation of ATR’s role in development and tumorigenesis is complicated by the lethality of homozygous knockout mice and the limited effects of heterozygous deficiency. To overcome this limitation, we sought to create mice with a hypomorphic Atr mutation based on the ATR mutation in the human disease Seckel syndrome-1 (SCKL1). Homozygous SCKL1 mice were generated by targeted knock-in of the A → G SCKL1 mutation. Western blot and RT-PCR analysis established that homozygotes have no reduction in Atr protein or increase in missplicing as is seen in humans. Thus, the A → G substitution alone is not sufficient to reproduce in mice the effects that are seen in humans. However, homozygous SCKL1 mice that retain the neo cassette used for targeting have an estimated 66-82% reduction in total Atr protein levels due to missplicing into the neo cassette. Under conditions of APH-induced replication stress, primary fibroblasts from homozygous mice displayed an increase in overall chromosome damage and an increase in gaps and breaks at specific common fragile sites. In addition, mutant cells display a significant delay in checkpoint induction and an increase in DNA damage as assayed by Chk1 phosphorylation and γ-H2ax levels, respectively. These mice provide a novel model system for studies of Atr deficiency and replication stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Freshwater crayfish invasions in South Africa: past,present and potential future

Freshwater crayfish invasions have been studied around the world, but less so in Africa, a continent devoid of native freshwater crayfish. The present study reviews historical and current information on alien freshwater crayfish species introduced into South Africa and aims to indicate which areas are at risk from invasion. As is the case elsewhere, South Africans have shown a keen interest in both farming and keeping freshwater crayfish as pets, which has resulted in Cherax cainii, Cherax destructor, Cherax quadricarinatus and Procambarus clarkii being introduced to the country. There is evidence of successful establishment in the wild for C. quadricarinatus and P. clarkii in different parts of the country. Species distribution models suggest that the eastern part of the country and parts of the Eastern and Western Cape are at higher risk of invasion. At present, illegal translocations represent the most likely pathway of crayfish spread in South Africa. A continued risk of invasion by freshwater crayfish species in South Africa is highlighted, which reinforces the need for more research, as well as for strong mitigation measures, such as stronger policing of existing regulations, management or eradication where feasible and public education.     

The PagN protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium is an adhesin and invasin

Background  

The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.     

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

Background  

Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.     

Skeletal abnormalities and extra-skeletal ossification in mice with restricted Gsα deletion caused by a renin promoter-Cre transgene

We have recently generated a transgenic mouse line (termed hRen-Cre) that expresses Cre-recombinase under the control of a 12.2-kb fragment of the human renin promoter. In the present study, we have crossed hRen-Cre mice with a mouse strain in which exon 1 of the Gnas gene is flanked by loxP sites. Gnas encodes the α-subunit of the stimulatory G protein (Gsα). Our aim has been to generate a mouse model with locally restricted inactivation of Gsα to extend studies of the role of Gsα function in vivo. Mice with local Cre-mediated inactivation of Gsα (rCre-Gsα) are viable and fertile. Their most obvious phenotype consists of marked skeletal malformations of the forelimbs in which computer-tomography scans reveal shortened and fused extremity bones. Extraskeletal ossifications occur in the subcutis and in skeletal muscles associated with the affected long bones. Plasma calcium, phosphate and parathyroid hormone are normal. Skin histology has demonstrated diffuse mineralization and ossification associated with the basal cells of hair follicles. This phenotype in part resembles syndromes in humans associated with loss-of-function of Gsα, such as Albright hereditary osteodystrophy and progressive osseous heteroplasia. The renal phenotype of rCre-Gsα mice is inconspicuous. Plasma renin concentration, ambient urine osmolarity, and the glomerular filtration rate of rCre-Gsα mice do not differ from controls. The absence of measurable functional changes in the renin-angiotensin system indicates insufficient Cre expression in juxtaglomerular granular cells in this strain of mice. Nevertheless, the present report reaffirms the importance of Gsα signaling for bone development and the suppression of ectopic ossification. This work was supported by intramural funds from the National Institute of Diabetes and Digestive and Kidney Diseases. H. Castrop was supported by a grant from the Deutsche Forschungsgemeinschaft (CA278/4-1).     

Salt effects on beta-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond beta-glucosidase. For cations (Cl(-) salts) the effectiveness follows the series: Cu(+2), Fe(+2)>Zn(+2)>Li(+)>Ca(+2)>Mg(+2)>Cs(+)>NH(4)(+)>Rb(+)>K(+)>Na(+) and for anions (Na(+) salts) the series is: I(-)>ClO(4)(-)>(-)SCN>Br(-) approximately NO(3)(-)>Cl(-) approximately (-)OAc>F(-) approximately SO(4)(-2). The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k(cat)/K(m) for the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK(a)s and not to an effect on the pH-independent second-order rate constant, (k(cat)/K(m))(lim). For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK(a1)increases (from 3.7 to 4.9) and the apparent pK(a2)decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k(cat)/K(m))(lim) (=9 x 10(4) M(-1) s(-1)) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK(a)s when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK(a) shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK(a) of 3.7 and dissociated from the group with a pK(a) of 7.2) is very small ( approximately 0.03%). At higher salt concentrations, where the two pK(a)s become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k(cat)/K(m))(lim), of the monoprotonated species. For other enzymes which may show such salt-induced pK(a) shifts, this provides a convenient test for the role of reverse protonation.     

Cell-associated hemolysis activity in the clinical strain of <Emphasis Type="Italic">Pseudomonas fluorescens MFN1032</Emphasis>

Background  

MFN1032 is a clinical Pseudomonas fluorescens strain able to grow at 37°C. MFN1032 cells induce necrosis and apoptosis in rat glial cells at this temperature. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides. Under laboratory conditions, this activity is not expressed at 37°C. This activity is tightly regulated and is subject to phase variation.