Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates

Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.     

Apolipoprotein B amino acid 3611 substitution from arginine to glutamine creates the Ag (h/i) epitope: the polymorphism is not associated with differences in serum cholesterol and apolipoprotein B levels

Summary A G-to A-DNA sequence change in exon 26 of the human apolipoprotein B (apo B) gene leads to a glutamine substitution for arginine at codon 3611 of the mature apolipoprotein B100 and causes a loss of an MspI site. In 106 Finnish individuals, a complete correspondence exists between this MspI polymorphic site and the Ag (h/i) immunochemical polymorphism. Linkage disequilibrium was found between this MspI polymorphic site and the apo B XbaI and EcoRI variable sites and the Ag (a1/d) and (c/g) epitope pairs; there is apparent linkage equilibrium with the apo B PvuII variable site. Based on three population studies (samples from London, Finland and Italy), no significant association was found between this RFLP and serum cholesterol and apo B levels. These data suggest that the arginine 3611glutamine 3611 substitution has no significant effect on apo B function.     

Genetic changes in a vendace Covegonus albula (L.) population, 92 years after introduction

The Osensjmn vendace, Coregonus albula (L.), population was founded in 1895 by introduction of about 90 000 fry from Lake Mjøsa. After 92 years, or about 46 generations of isolation, remarkable genetic changes were evident among the transplants compared to their source population. Electrophoresis of 33 enzyme loci revealed that allele frequencies differed significantly in eight of the 10 polymorphic loci. Nei's unbiased genetic distance was D = 0.012, representing the level of population differentiation usually observed among distant watercourses in vendace. Genetic variability was clearly higher among the transplants: average observed heterozygosity was 14.8% compared with 11.2% among Lake Mjøsa vendace. The high genetic variation among Osensjoen vendace shows that the divergence is not due to a small number of founders or bottlenecks in later generations. Selection by the contrasting environmental conditions between the habitats of the donor and transplanted populations is the most plausible hypothesis to explain the genetic changes in the Osensjsen vendace.     

Reduction of genetic variability in a hatchery stock of brown trout, Salmo trutta L.

Enzyme gene variability in a lake-dwelling brown trout, Salmo Irulla , population was compared with its hatchery derivative by starch gel electrophoresis. A loss of genetic variability was detected in the hatchery stock founded 16 years ago in comparison to the present day wild stock; of seven originally polymorphic loci ( Aat-4, Ck-l, Gpi-3, G3p-2, Ldh-1, Mclh-2 and Pgm-l ) two, Gpi-3 and Ldh-1 , had become monomorphic. The overall hetero-zygosity was reduced by a third from 6–2% to 4–2%. A comparison between three successive hatchery cohorts indicates a distinction between stock heterogeneity. The decrease in genetic variability is probably attributable to small population size in the founding event with subsequent genetic drift and inadvertent selection during hatchery procedures.     

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Modulation of GABAergic neurotransmission in the brain by dipeptides

The effects of endogenous and synthetic peptides containing GABA or its analogues on the GABA/benzodiazepine/chloride ionophore, complex, GABA_B receptor, Cl fluxes, GABA release and GABA uptake were studied using synaptic membranes, crude synaptoneurosomal preparations and slices prepared from the rat and mouse brain. The sodium-independent binding of GABA was strongly inhibited by GABA-histidine, followed by -glutamyl-homotaurine, GABA-glycine and -glutamyl-GABA. The binding of diazepam was slightly enhanced by the same peptides. The peptides alone had no effect on the chloride fluxes, but GABA-histidine, -glutamyl-GABA and GABA-glycine enhanced while -glutamyl-homotaurine and GABA-taurine inhibited GABA-stimulated chloride uptake. GABA-histidine was the most effective displacer of baclofen binding, but -glutamyl-homotaurine was entirely ineffective. The uptake of GABA was markedly inhibited in synaptosomal preparations by GABA-histidine, while all other peptides were less effective. -Glutamyl-taurine attenuated but -glutamyl-homotaurine and GABA-glycine enhanced the potassium-stimulated release of GABA. The present actions of GABA-histidine in vitro may be of significance for GABAergic neurotransmission in vivo.     

Helicobacter infection induces podosome assembly in primary hepatocytes in vitro

Helicobacter pylori (H. pylori) infection may contribute to many extragastric diseases including liver cirrhosis and hepatocellular carcinoma. However, the exact mechanism by which H. pylori induces the liver damage is largely unknown. We used cultured mouse primary hepatocytes as an in vitro model to investigate different aspects of liver physiology and pathology. In this study, we show that primary hepatocytes are able to assemble actin-based cytoskeletal structures called podosomes at the ventral plasma membrane. These structures are positive for podosome markers such as cortactin, vinculin and integrins and comprise proteolytic potential. Infection with the pathogen H. pylori further stimulates the formation of podosomes in primary hepatocytes. The use of pharmacological inhibitors reveals that this response is mediated, at least in part, by TGFβ, a cytokine known to regulate podosome formation in endothelial cells. Similar results are obtained with the hepatoma cell line Huh7. Podosome formation is associated with increased hepatocyte degrading capacities but also with reduced cell motility. Therefore, podosome assembly translates into hepatocyte malfunction. Our study supports the hypothesis that hepatocytes can also assemble podosomes under pathological conditions in vivo.     

Women With Hypertensive Pregnancies Have Difficulties in Regaining Pre‐pregnancy Weight and Show Metabolic Disturbances

The aim was to determine maternal weight gain and body composition during pregnancy and 3 months postpartum in women with uncomplicated singleton and twin pregnancies and in women with gestational diabetes (GDM) and gestational hypertension (GH). This prospective study includes four groups of subjects: those with an uncomplicated pregnancy (n = 32), those with a diagnosis of GH (n = 28), those with a diagnosis of GDM (n = 52), and those with twin pregnancy (n = 11). Their body compositions were estimated by a bioimpedance analysis and fasting lipids and glucose levels were determined during the pregnancy and 3 months after pregnancy. Women with GDM were 11.7 kg heavier than the reference group before pregnancy, whereas weight before pregnancy was not different in other investigated groups. Weight loss after delivery was attenuated in GH group. Percentage body fat remained elevated in women with GDM (34.1 ± 7.0%) and hypertension (31.5 ± 6.4%) at 3 months after pregnancy. Also their total cholesterol and low‐density lipoprotein (LDL)‐cholesterol levels as well as fasting glucose remained elevated in comparison to values of the reference group. In conclusion, women with hypertensive pregnancies, though not overweight before pregnancy, gain and retain excess gestational weight and this leads to metabolic abnormalities similar to those seen in women GDM. Thus, postpartum period appears to be critical for weight management and interventional programs are called for.     

Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1

Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as a result of attachment of alpha(2)I. A molecular model, constructed on the basis of the EV1-integrin complex, shows that multiple alpha(2)beta(1) heterodimers can bind at adjacent sites around the virus 5-fold symmetry axes without steric hindrance. In agreement with this, virus attachment to alpha(2)beta(1) integrin on the cell surface was found to result in integrin clustering, which can give rise to signaling and facilitate the initiation of the viral entry process that takes place via caveolae-mediated endocytosis.