Increased levels of superoxide in brains from old female rats

Hypertension, aging and a range of neurodegenerative diseases are associated with increased oxidative damage. The present study examined whether superoxide (O₂^•-) levels in brain are increased during aging in female rats, and the role of superoxide dismutase (SOD) and oestrogen in regulating O₂^•- levels.

Young adult (3 month) and old (11 month) female spontaneously hypertensive stroke prone rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied. O₂^•- levels were measured in brain homogenates by lucigenin chemiluminescence and SOD expression by Western blotting. Ageing significantly increased brain O₂^•- levels in WKY (cortex +216%, hippocampus +320%, striatum +225%) and to a greater extent in SHRSP (cortex +540%, hippocampus +580%, striatum +533%). Older SHRSP showed a decline in cortical Cu/Zn SOD expression compared to young adult SHRSP. Oestrogen did not attenuate O₂^•- levels.

The results show a significant age-dependent increase in brain O₂^•- levels which is exaggerated in SHRSP. The excess cortical O₂^•- levels in the SHRSP may be associated with a down-regulation of Cu/Zn SOD but are not related to a decrease in oestrogen.     

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.     

Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats

Estrogen treatment has been shown to reduce ischemic brain damage. Because endogenous estrogen levels fluctuate markedly during the estrous cycle, we investigated the effect of stage of estrous cycle on ischemic brain damage. Halothane anesthetized 3- to 5-mo-old female Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) in proestrus (high estradiol levels) or metestrus (low estradiol levels) underwent permanent middle cerebral artery occlusion. In SHRSP, infarct volume at 24 h postocclusion was 24% smaller in proestrus compared with metestrus [208.6 +/- 9.5 mm(3) (n = 7) vs. 272.7 +/- 23.8 mm(3) (n = 7), respectively, means +/- SE; P = 0.0278, unpaired t-test]. In WKY, infarct volumes were similar in proestrus and metestrus [157.0 +/- 5.4 mm(3) (n = 5) and 131.5 +/- 16.5 mm(3) (n = 8), respectively; P = not significant (NS)]. Brain swelling (ipsilateral minus contralateral hemispheric volumes) was similar in proestrus and metestrus for SHRSP [138 +/- 9 mm(3) (n = 6) and 136 +/- 10 mm(3) (n = 7), respectively] and for WKY [103 +/- 15 mm(3) (n = 5) and 90 +/- 11 mm(3) (n = 8), respectively]. Thus the reduction in infarct size in SHRSP is caused by a true attenuation of the infarct volume and not simply by a reduction in brain edema.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

Integrin-alphavbeta6, a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.

Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.     

Structural characterization of NETNES, a novel glycoconjugate in Trypanosoma cruzi epimastigotes

The unicellular stercorarian protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. The epimastigote form of the parasite is covered in a dense coat of glycoinositol phospholipids and short glycosylphosphatidylinositol (GPI)-anchored mucinlike molecules. Here, we describe the purification and structural characterization of NETNES, a relatively minor but unusually complex glycoprotein that coexists with these major surface components. The mature glycoprotein is only 13 amino acids in length, with the sequence AQENETNESGSID, and exists in two forms with either four or five post-translational modifications. These are either one or two asparagine-linked oligomannose glycans, two linear alpha-mannose glycans linked to serine residues via phosphodiester linkages, and a GPI membrane anchor attached to the C-terminal aspartic acid residue. The variety and density of post-translational modifications on an unusually small peptide core make NETNES a unique type of glycoprotein. The N-glycans are predominantly Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn; the phosphate-linked glycans are a mixture of (Manalpha1-2)0-3Man1-P-Ser; and the GPI anchor has the structure Manalpha1-2(ethanolamine phosphate)Manalpha1-2Manalpha1-6Manalpha1-4(2-aminoethylphosphonate-6)GlcNalpha1-6-myo-inositol-1-P-3(sn-1-O-(C16:0)alkyl-2-O-(C16:0)acylglycerol). Four putative NETNES genes were found in the T. cruzi genome data base. These genes are predicted to encode 65-amino acid proteins with cleavable 26-amino acid N-terminal signal peptides and 26-amino acid C-terminal GPI addition signal peptides.     

The effects of selection,drift and genetic variation on life‐history trait divergence among insular populations of natterjack toad,Bufo calamita

Although loss of genetic variation is frequently assumed to be associated with loss of adaptive potential, only few studies have examined adaptation in populations with little genetic variation. On the Swedish west coast, the northern fringe populations of the natterjack toad Bufo calamita inhabit an atypical habitat consisting of offshore rock islands. There are strong among‐population differences in the amount of neutral genetic variation, making this system suitable for studies on mechanisms of trait divergence along a gradient of within‐population genetic variation. In this study, we examined the mechanisms of population divergence using Q_ST–F_ST comparisons and correlations between quantitative and neutral genetic variation. Our results suggest drift or weak stabilizing selection across the six populations included in this study, as indicated by low Q_ST–F_ST values, lack of significant population × temperature interactions and lack of significant differences among the islands in breeding pond size. The six populations included in this study differed in both neutral and quantitative genetic variation. Also, the correlations between neutral and quantitative genetic variation tended to be positive, however, the relatively small number of populations prevents any strong conclusions based on these correlations. Contrary to the majority of Q_ST–F_ST comparisons, our results suggest drift or weak stabilizing selection across the examined populations. Furthermore, the low heritability of fitness‐related traits may limit evolutionary responses in some of the populations.