Obesity-inducing lesions of the central nervous system alter leptin uptake by the blood-brain barrier.

Leptin regulates body adiposity by decreasing feeding and increasing thermogenesis. Obese humans and some obese rodents are resistant to peripherally administered leptin, suggesting a defect in the transport of leptin across the blood-brain barrier (BBB). Defective transport of exogenous leptin occurs in some models of obesity, but in other models transport is normal. This shows that factors other than obesity are associated with impairment of leptin transport across the BBB. In order to further investigate these factors, we determined leptin transport in rats made obese by lesioning of the ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), or posterodorsal amygdala (PDA). These regions all contain leptin receptors and lesions there induce obesity and hyperleptinemia and alter the levels of many feeding hormones which might participate in leptin transporter regulation. We measured the uptake of radioactively labeled leptin by the BBB by multiple-time regression analysis which divides uptake into a reversible phase (Vi, e.g., receptor/transporter binding to the brain endothelial cell) and an irreversible phase (Ki, complete transport across the BBB). Leptin uptake was not affected in rats with VMH lesions. No significant change occurred in the entry rate (Ki) for any group, although Ki declined by over 35% in rats with PVN lesions. Decreased uptake was observed in rats with PVN lesions and with PDA lesions. This was primarily due to a reduced Vi (about 21% for the PDA). This decreased uptake is most likely explained by decreased binding of leptin to the brain endothelial cell, which could be because of decreased binding by either receptors or transporters. This suggests that some of the feeding hormones controlled by the PVN and PDA may participate in regulating leptin uptake by the BBB.     

Localization of deletion breakpoints in radiation-induced mutants of the hprt gene in hamster cells

DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiations and others occurring naturally, to identify those with large alterations in part of the gene. DNA from these mutants was restricted further with different endonucleases and probed to establish the patterns of restriction fragments remaining. Of 15 mutants characterized, one showed a duplication of part of the 5' end of the gene, and the remainder showed deletions of various sizes. It was possible to approximately locate the breakpoints of the deletions by comparison of fragment patterns to a recently-established map of the hamster gene. The relatively small number of mutants examined precludes rigorous analysis of the distribution of breakpoints in the hprt gene, but taken with other recent studies of deletion mutagenesis it is suggested that non-random induction or selection of this type of mutation may occur.     

Xeromammography in the Early Detection of Breast Cancer: Community Hospital Experience and Approach

Community hospitals can do much in the general effort toward earlier detection of breast cancer. Using xeromammography in the department of general radiology of one community hospital, 21 cases of occult carcinoma were detected in 2,392 patients in a two year period. Patients were both symptomatic and asymptomatic. This percentage is comparable to results in previously published series of similar patient populations, and can be expected to be slightly higher than screening populations of totally asymptomatic patients. In 24 percent of cases of occult carcinoma there was axillary node involvement, compared with 42 percent axillary node involvement in cases of nonoccult carcinoma.Early detection efforts are currently centered on improving thoroughness in physical examinations, stressing breast self-examination and identifying patients in high-risk categories. These, together with proper periodic use of mammograms, will yield the highest rate of early carcinoma detection until more sensitive biologic markers are developed.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

2-Naphthoate catabolic pathway in Burkholderia strain JT 1500.

Burkholderia strain (JT 1500), able to use 2-naphthoate as the sole source of carbon, was isolated from soil. On the basis of growth characteristics, oxygen uptake experiments, enzyme assays, and detection of intermediates, a degradation pathway of 2-naphthoate is proposed. The features of this pathway are convergent with those for phenanthrene. We propose a pathway for the conversion of 2-naphthoate to 1 mol (each) of pyruvate, succinate, and acetyl coenzyme A and 2 mol of CO2. During growth in the presence of 2-naphthoate, six metabolites were detected by thin-layer chromatography, high-performance liquid chromatography, and spectroscopy. 1-Hydroxy-2-naphthoate accumulated in the culture broth during growth on 2-naphthoate. Also, the formation of 2'-carboxybenzalpyruvate, phthalaldehydate, phthalate, protocatechuate, and beta-carboxy-cis,cis-muconic acid was demonstrated. (1R,2S)-cis-1,2-Dihydro-1,2-dihydroxy-2-naphthoate was thus considered an intermediate between 2-naphthoate and 1-hydroxy-2-naphthoate, but it was not transformed by whole cells or their extracts. We conclude that this diol is not responsible for the formation of 1-hydroxy-2-naphthoate from 2-naphthoate but that one of the other three diastereomers is not eliminated as a potential intermediate for a dehydration reaction.     

Therapy with minocycline aggravates experimental rabies in mice

Minocycline is a tetracycline derivative with antiapoptotic and anti-inflammatory properties, and the drug has been shown to have beneficial effects in a variety of models of neurological disorders. The potentially neuroprotective role of minocycline was assessed in experimental in vitro and in vivo models of rabies virus infection. In this study, 5 nM minocycline did not improve the viability of embryonic mouse cortical and hippocampal neurons infected in vitro with the attenuated SAD-D29 strain of rabies virus, based on assessments using trypan blue exclusion. Two-day-old ICR mice were inoculated in the right hind limb thigh muscle with SAD-D29, and they received daily subcutaneous injections of either 50 mg/kg minocycline or vehicle (phosphate-buffered saline). Infected minocycline-treated mice experienced an earlier onset of neurologic signs and greater mortality (83% versus 50%) than those receiving vehicle (log rank test, P=0.002 and P=0.003, respectively). Immunohistochemical analysis of rabies virus antigen distribution was performed at early time points and in moribund mice. There were greater numbers of infected neurons in the regional brain areas of minocycline-treated mice than in vehicle-treated mice, which was significant in the CA1 region of the hippocampus. There was less apoptosis (P=0.01) and caspase 3 immunostaining (P=0.0008) in the midbrains of mice treated with minocycline than in mice treated with vehicle, consistent with a neuroprotective role of neuronal apoptosis that may have had a mild effect of inhibiting viral spread. Reduced infiltration of CD3+ T cells was observed in the pons/medulla of moribund mice that received minocycline therapy (P=0.008), suggesting that the anti-inflammatory actions of minocycline may intensify the neurologic disease. These findings indicate that minocycline has important detrimental effects in the therapy of experimental rabies. Empirical therapy with minocycline should therefore be approached with caution in cases of human rabies and possibly other viral encephalitides until more experimental data become available.     

Rangewide phylogeography in the greater horseshoe bat inferred from microsatellites: implications for population history, taxonomy and conservation

The distribution of genetic variability across a species' range can provide valuable insights into colonization history. To assess the relative importance of European and Asian refugia in shaping current levels of genetic variation in the greater horseshoe bats, we applied a microsatellite-based approach to data collected from 56 localities ranging from the UK to Japan. A decline in allelic richness from west Asia to the UK and analyses of F(ST) both imply a northwestward colonization across Europe. However, sharp discontinuities in gene frequencies within Europe and between the Balkans and west Asia (Syria/Russia) are consistent with suture zones following expansion from multiple refugia, and a lack of recent gene flow from Asia Minor. Together, these results suggest European populations originated from west Asia in the ancient past, and experienced a more recent range expansion since the Last Glacial Maximum. Current populations in central Europe appear to originate from the Balkans and those from west Europe from either Iberia and/or Italy. Comparisons of R(ST )and F(ST) suggest that stepwise mutation has contributed to differentiation between island and continental populations (France/UK and China/Japan) and also among distant samples. However, pairwise R(ST) values between distant populations appear to be unreliable, probably due to size homoplasy. Our findings also highlight two priorities for conservation. First, stronger genetic subdivision within the UK than across 4000 km of continental Eurasia is most likely the result of population fragmentation and highlights the need to maintain gene flow in this species. Second, deep splits within China and between Europe and China are indicative of cryptic taxonomic divisions which need further investigation.     

Basal defenses induced in pepper by lipopolysaccharides are suppressed by Xanthomonas campestris pv. vesicatoria

The nonpathogenic hrcC mutant of Xanthomonas campestris pv. vesicatoria 85-10::hrpA22 multiplied in pepper leaves if it was mixed with pathogenic strains of X. campestris pv. vesicatoria. Reactions to the mutant alone included localized deposition of phenolics and callose in papillae, and alterations to the plant cell wall leading to increased electron density. Electron microscopy showed that the localized responses were suppressed in the presence of wild-type bacteria but other wall changes occurred at some sites, involving cellulose-rich ingrowth of the wall. Multiplication of the hrp mutant in mixed inocula was confirmed by tagging 85-10::hrpA22 using immunocytochemical location of AvrBs3 expressed from the plasmid pD36. Elicitors of callose deposition and other wall changes were isolated from the hrcC mutant. Activity in extracts of bacteria was attributed to the presence of high molecular weight lipopolysaccharides (LPS). Wild-type X. campestris pv. vesicatoria suppressed induction of structural changes caused by purified LPS. Results obtained suggest that effector proteins produced by phytopathogenic bacteria and delivered by the type III secretion system may have a key role in suppressing the basal defense responses activated by bacterial LPS, which lead to restricted multiplication of nonpathogens such as hrp mutants.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Design and synthesis of 4-[3,5-dioxo-11-oxa-4,9-diazatricyclo[5.3.1.02,6]undec-4-yl]-2-trifluoromethyl-benzonitriles as androgen receptor antagonists

A novel series of 4-[3,5-dioxo-11-oxa-4,9-diazatricyclo[5.3.1.0^2,6]undec-4-yl]-2-trifluoromethyl-benzonitriles has been synthesized. The ability of these compounds to act as antagonists of the androgen receptor was investigated and several were found to have potent activity in vitro and in vivo.