Effects of streptozotocin-induced diabetes on fetal development of the rat

The purpose of this study was to determine whether or not in rats with experimentally induced diabetes there is an increased frequency of congenital malformations; data in the literature are not consistent on this point. Virgin CD females rats were injected with 40-50 mg/kg streptozotocin (Stz) before mating (SIBM group) or on the first day of pregnancy (SI1). Both SIBM and SI1 females were divided into two groups according to their blood glucose levels: severely diabetic (SD, greater than 300 mg%) and mildly diabetic (MD, 120-250 mg%). Food and water consumption by the control and MD groups were the same, but the SD females developed polyphagia, polyuria, and polydypsia, which continued to increase throughout pregnancy, as did the blood glucose levels. All the MD females mated and carried to term. In SD females both frequency of mating and fertility were only slightly lower than in the controls. All the females were killed on the 21st day of pregnancy. Pre- and postimplantation losses were the same for diabetic and control rats, but SIBM-SD females ovulated less than other groups. Weights of fetuses of SD dams were lower and blood sugar levels higher than those of the other groups. The placentas of SD rats were significantly heavier and there was cystic degeneration of spongiosa. The incidence of major malformations was minimal (approximately 2%) in fetuses of SD females and there were none at all in controls or MD females. In conclusion, our data are in agreement with those of other investigators who have found that rats with experimentally induced diabetes have smaller fetuses and increased placental weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebral Blood Volume ASPECTS Is the Best Predictor of Clinical Outcome in Acute Ischemic Stroke: A Retrospective,Combined Semi-Quantitative and Quantitative Assessment

Introduction

The capability of CT perfusion (CTP) Alberta Stroke Program Early CT Score (ASPECTS) to predict outcome and identify ischemia severity in acute ischemic stroke (AIS) patients is still questioned.

Methods

62 patients with AIS were imaged within 8 hours of symptom onset by non-contrast CT, CT angiography and CTP scans at admission and 24 hours. CTP ASPECTS was calculated on the affected hemisphere using cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) maps by subtracting 1 point for any abnormalities visually detected or measured within multiple cortical circular regions of interest according to previously established thresholds. MTT-CBV ASPECTS was considered as CTP ASPECTS mismatch. Hemorrhagic transformation (HT), recanalization status and reperfusion grade at 24 hours, final infarct volume at 7 days and modified Rankin scale (mRS) at 3 months after onset were recorded.

Results

Semi-quantitative and quantitative CTP ASPECTS were highly correlated (p<0.00001). CBF, CBV and MTT ASPECTS were higher in patients with no HT and mRS≤2 and inversely associated with final infarct volume and mRS (p values: from p<0.05 to p<0.00001). CTP ASPECTS mismatch was slightly associated with radiological and clinical outcomes (p values: from p<0.05 to p<0.02) only if evaluated quantitatively. A CBV ASPECTS of 9 was the optimal semi-quantitative value for predicting outcome.

Conclusions

Our findings suggest that visual inspection of CTP ASPECTS recognizes infarct and ischemic absolute values. Semi-quantitative CBV ASPECTS, but not CTP ASPECTS mismatch, represents a strong prognostic indicator, implying that core extent is the main determinant of outcome, irrespective of penumbra size.     

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an Enterovirus of the Picornaviridae family, uses the complement regulator decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF consisting of short consensus repeat domains 3 and 4 from cryo-negative stain electron microscopy data (EMD code 1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the 2-fold symmetry axes. Despite this general similarity our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF(34) and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB code 1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF binding phenotype in these viruses.     

Lethal and sublethal effects of long‐lasting insecticide‐treated nets on the invasive bug Halyomorpha halys

Halyomorpha halys is a polyphagous insect species with an original eastern Asiatic distribution, which was recently and accidentally introduced in the USA and Europe, where it became a serious agricultural pest. Chemicals have been widely used for its control leading often to failure of IPM programmes. Several approaches aimed at pest monitoring and control are currently under investigation, for example trapping, screening, border management and biological control. In the present work, we investigated the lethal and sublethal effects of the use of a long‐lasting insecticide‐treated net (LLIN with α‐cypermethrin), focusing on the perspective to control H. halys in an integrated approach. All experiments were carried out in the laboratory either in small arenas, at five exposure times (5, 15, 30, 45 and 60 min) or in large cages at 8 hr exposure. In small arenas, the LLIN induced sublethal effects and/or effectively killed the adults. A higher adult mortality was observed after longer exposure times (LT₉₀ was 51.64 min for females and 40.83 min for males). However, several specimens recovered from sublethal effects, with higher recovery rates after shorter exposure times. In the cage experiments, a significantly higher mortality (65% males and 75% of females) was recorded compared with controls. LLINs are physical barriers that can improve crop protection due to their insecticidal activity, and can be reasonably applied in various attract‐and‐kill systems both in glasshouses and in the field.     

The Escherichia coli Cell Division Protein and Model Tat Substrate SufI (FtsP) Localizes to the Septal Ring and Has a Multicopper Oxidase-Like Structure

The Escherichia coli protein SufI (FtsP) has recently been proposed to be a component of the cell division apparatus. The SufI protein is also in widespread experimental use as a model substrate in studies of the Tat (twin arginine translocation) protein transport system. We have used SufI-GFP (green fluorescent protein) fusions to show that SufI localizes to the septal ring in the dividing cell. We have also determined the structure of SufI by X-ray crystallography to a resolution of 1.9 Å. SufI is structurally related to the multicopper oxidase superfamily but lacks metal cofactors. The structure of SufI suggests it serves a scaffolding rather than an enzymatic role in the septal ring and reveals regions of the protein likely to be involved in the protein-protein interactions required to assemble SufI at the septal ring.     

Shigella flexneri Spa15 crystal structure verified in solution by double electron electron resonance

Shigella flexneri Spa15 is a chaperone of the type 3 secretion system, which binds a number of effectors to ensure their stabilization prior to secretion. One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG. In this study, Spa15 alone and in complex with IpgB1 has been studied by double electron electron resonance, an experiment that gives distance information showing the spacial separation of attached spin labels. This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets. The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.     

Identification and characterization of the ‘missing’ terminal enzyme for siroheme biosynthesis in α‐proteobacteria

It has recently been shown that the biosynthetic route for both the d₁‐haem cofactor of dissimilatory cd₁ nitrite reductases and haem, via the novel alternative‐haem‐synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d₁‐haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild‐type phenotype of an Escherichia coli mutant‐strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α‐proteobacteria as the terminal enzyme of siroheme biosynthesis.     

Rapid-cooling and storage of plant nematodes at -140 degrees C

Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.     

Functional and structural characterization of the integrase from the prototype foamy virus

Establishment of the stable provirus is an essential step in retroviral replication, orchestrated by integrase (IN), a virus-derived enzyme. Until now, available structural information was limited to the INs of human immunodeficiency virus type 1 (HIV-1), avian sarcoma virus (ASV) and their close orthologs from the Lentivirus and Alpharetrovirus genera. Here, we characterized the in vitro activity of the prototype foamy virus (PFV) IN from the Spumavirus genus and determined the three-dimensional structure of its catalytic core domain (CCD). Recombinant PFV IN displayed robust and almost exclusively concerted integration activity in vitro utilizing donor DNA substrates as short as 16 bp, underscoring its significance as a model for detailed structural studies. Comparison of the HIV-1, ASV and PFV CCD structures highlighted both conserved as well as unique structural features such as organization of the active site and the putative host factor binding face. Despite possessing very limited sequence identity to its HIV counterpart, PFV IN was sensitive to HIV IN strand transfer inhibitors, suggesting that this class of inhibitors target the most conserved features of retroviral IN-DNA complexes.     

Structure of bacterial 3beta/17beta-hydroxysteroid dehydrogenase at 1.2 A resolution: a model for multiple steroid recognition

The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Comamonas testosteroni. It catalyzes the reversible reduction/dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids. Crystallographic analysis at 1.2 A resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry. This is one of the largest oligomeric structures refined at this resolution. The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices. The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR). Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism. In contrast to other SDR apostructures determined, the substrate binding loop is well-defined. Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.