Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.

Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 µM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

Mechanisms of extracellular S0 globule production and degradation in Chlorobaculumtepidum via dynamic cell-globule interactions.

The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.

Primate reaching cued by multichannel spatiotemporal cortical microstimulation.

Both humans and animals can discriminate signals delivered to sensory areas of their brains using electrical microstimulation. This opens the possibility of creating an artificial sensory channel that could be implemented in neuroprosthetic devices. Although microstimulation delivered through multiple implanted electrodes could be beneficial for this purpose, appropriate microstimulation protocols have not been developed. Here, we report a series of experiments in which owl monkeys performed reaching movements guided by spatiotemporal patterns of cortical microstimulation delivered to primary somatosensory cortex through chronically implanted multielectrode arrays. The monkeys learned to discriminate microstimulation patterns, and their ability to learn new patterns and new behavioral rules improved during several months of testing. Significantly, information was conveyed to the brain through the interplay of microstimulation patterns delivered to multiple electrodes and the temporal order in which these electrodes were stimulated. This suggests multichannel microstimulation as a viable means of sensorizing neural prostheses.

Identifying barriers to prehospital thrombolysis in the treatment of acute myocardial infarction.

BACKGROUND AND OBJECTIVES: After an acute myocardial infarction (AMI) prehospital thrombolysis (PHT) reduces mortality compared with inhospital thrombolysis. In practice, a relatively small proportion of the total population with AMI receives PHT. This study was designed to identify the current barriers to PHT. METHODS: A retrospective practice review of 57 consecutive patients treated in or before arrival at a district general hospital emergency department. All patients received thrombolysis for an AMI. RESULTS: The main barriers to delivery of PHT appear to be the inclusion and exclusion criteria laid out in the ambulance service central guidelines. Despite recent widening of the inclusion criteria, 54% of patients eligible for immediate treatment on arrival in hospital either received or were eligible for PHT. CONCLUSION: To increase the number of patients who are eligible for PHT these guidelines need to be revised further in line with inhospital criteria for thrombolysis.

Localization and functional role of the calmodulin-binding domain of phospholamban in cardiac sarcoplasmic reticulum vesicles.

Limited proteolysis and affinity-labeling techniques have been used to localize the calmodulin-binding domain of phospholamban, the major substrate for both cAMP- and calmodulin-dependent protein kinases in cardiac sarcoplasmic reticulum (SR). SR vesicles, treated with increasing concentrations of trypsin (likely hydrolyzing at Arg-25 in the cytoplasmic region of phospholamban), exhibited a subsequent loss of both cAMP- and calmodulin-dependent phosphorylation, as well as calmodulin affinity-labeling of phospholamban. When SR vesicles were treated with increasing concentrations of chymotrypsin (which likely cleaves at Tyr-6 of phospholamban) there was no effect on the cAMP-dependent phosphorylation of phospholamban. However, similar concentrations of chymotrypsin resulted in a loss of both calmodulin affinity-labeling and calmodulin-dependent phosphorylation of phospholamban (at Thr-17). When SR vesicles were treated with increasing concentrations of Endoproteinase Lys-C (which hydrolyzes phospholamban at Lys-3) both the calmodulin affinity-labeling and the calmodulin-dependent, but not the cAMP-dependent, phosphorylation of phospholamban were inhibited. These data were complemented by 1H-NMR studies on the complex formed by calmodulin and a phospholamban peptide. These data suggest that binding of calmodulin to phospholamban may be an essential intermediate step in the calmodulin-dependent phosphorylation of phospholamban.

Predicting the probability of abortion in dairy cows: a hierarchical Bayesian logistic-survival model using sequential pregnancy data.

Although abortion contributes substantially to poor reproductive health of dairy herds, little is known about the predictability of abortion based on age, previous abortion or gravidity (number of previous pregnancies). A poor understanding of effects of maternal factors on abortion risk exists, in part, because of methodological difficulties related to non-independence of multiple pregnancies of the same cow in analysis of fetal survival data. We prospectively examined sequential pregnancies to investigate relationships between fetal survival and putative dam risk factors for 2991 abortions from 24,706 pregnancies of 13,145 cows in nine California dairy herds. Relative risks and predicted probabilities of abortion (PPA) were estimated using a previously described hierarchical Bayesian logistic-survival model generalized to incorporate longitudinal data of multiple pregnancies from a single cow. The PPA increased with increasing dam age at conception, with increasing number of previous abortions, and if the previous pregnancy was aborted >60 days in gestation. The PPA decreased with increasing gravidity and with increasing number of days open. For cows that aborted, the median time to fetal death decreased slightly as gravidity increased. The study considers several methodological issues faced in epidemiologic investigations of fetal health, including multi-modal hazard functions, extensive censoring and non-independence of multiple pregnancies. The model improves our ability to predict bovine abortion and to characterize fetal survival, which have important applications to herd health management.

Comparison of spin-echo and gradient-echo T1-weighted and spin-echo T2-weighted images at 3T in evaluating term-neonatal myelination.

SUMMARY: A prior clinical report of 3T MR imaging in subsequently healthy very premature neonates imaged at term-equivalent age found that both gradient recalled-echo-T1WI and spin-echo-T2WI showed higher rates of myelinated structures, compared with spin-echo-T1WI. The current study set out to assess those rates on the same sequences at 3T in term neonates and thus consisted of 16 term neonates with normal-appearing MR imaging findings who subsequently had normal findings at clinical follow-up. Two neuroradiologists independently assessed 19 structures in those infants on all 3 sequences. Gradient recalled-echo-T1WI showed a slightly higher rate of myelination (57.2%-72.4% of all structures) and interobserver agreement (κ = 0.546, P < .0001) than spin-echo-T2WI (58.2%-64.8%; κ = 0.468, P < .0001), while spin-echo-T1WI had the lowest myelination rate and agreement (25.0%-48.4%; κ = 0.384, P < .0001). Both observers noted that the following structures were myelinated in 88%-100% of patients on gradient recalled-echo-T1WI: the brachium of the inferior colliculus, decussation of the superior cerebellar peduncle, habenular commissure, medial lemniscus, pyramidal decussation, posterior limb of the internal capsule, and superior cerebellar peduncle; on spin-echo-T2WI, there was myelination in 88%-100% of the following structures: the brachium of the inferior colliculus, decussation of the superior cerebellar peduncle, inferior cerebellar peduncle, medial lemniscus, and posterior limb of the internal capsule. In conclusion, this study confirmed that similar to the findings in term-equivalent-age premature infants, myelination changes in term neonates may be best assessed on both gradient recalled-echo-T1WI and spin-echo-T2WI at 3T, and not on spin-echo-T1WI.

Strategies for optical control and simultaneous electrical readout of extended cortical circuits.

BACKGROUND: To dissect the intricate workings of neural circuits, it is essential to gain precise control over subsets of neurons while retaining the ability to monitor larger-scale circuit dynamics. This requires the ability to both evoke and record neural activity simultaneously with high spatial and temporal resolution. NEW METHOD: In this paper we present approaches that address this need by combining micro-electrocorticography (µECoG) with optogenetics in ways that avoid photovoltaic artifacts. RESULTS: We demonstrate that variations of this approach are broadly applicable across three commonly studied mammalian species - mouse, rat, and macaque monkey - and that the recorded µECoG signal shows complex spectral and spatio-temporal patterns in response to optical stimulation. COMPARISON WITH EXISTING METHODS: While optogenetics provides the ability to excite or inhibit neural subpopulations in a targeted fashion, large-scale recording of resulting neural activity remains challenging. Recent advances in optical physiology, such as genetically encoded Ca(2+) indicators, are promising but currently do not allow simultaneous recordings from extended cortical areas due to limitations in optical imaging hardware. CONCLUSIONS: We demonstrate techniques for the large-scale simultaneous interrogation of cortical circuits in three commonly used mammalian species.

Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of food intake and total body fat.

Pineal melatonin secretion declines with aging, whereas visceral fat, plasma insulin, and plasma leptin tend to increase. We have previously demonstrated that daily melatonin administration at middle age suppressed male rat intraabdominal visceral fat, plasma leptin, and plasma insulin to youthful levels; the current study was designed to begin investigating mechanisms that mediate these responses. Melatonin (0.4 microg/ml) or vehicle was administered in the drinking water of 10-month-old male Sprague Dawley rats (18/treatment) for 12 weeks. Half (9/treatment) were then killed, and the other half were submitted to cross-over treatment for an additional 12 weeks. Twelve weeks of melatonin treatment decreased (P<0.05) body weight (BW; by 7% relative to controls), relative intraabdominal adiposity (by 16%), plasma leptin (by 33%), and plasma insulin (by 25%) while increasing (P<0.05) locomotor activity (by 19%), core body temperature (by 0.5 C), and morning plasma corticosterone (by 154%), restoring each of these parameters toward more youthful levels. Food intake and total body fat were not changed by melatonin treatment. Melatonin-treated rats that were then crossed over to control treatment for a further 12 weeks gained BW, whereas control rats that were crossed to melatonin treatment lost BW, but food intake did not change in either group. Feed efficiency (grams of BW change per g cumulative food intake), a measure of metabolic function, was negative in melatonin-treated rats and positive in control rats before cross-over (P<0.001); this relationship was reversed after cross-over (P<0.001). Thus, melatonin treatment in middle age decreased BW, intraabdominal adiposity, plasma insulin, and plasma leptin, without altering food intake or total adiposity. These results suggest that the decrease in endogenous melatonin with aging may alter metabolism and physical activity, resulting in increased BW, visceral adiposity, and associated detrimental metabolic consequences.

Primary hyperparathyroidism presenting as anticonvulsant-induced osteomalacia.

A patient presented with the classic features of anticonvulsant-induced osteomalacia. Following discontinuance of diphenylhydantoin therapy and repletion with physiologic quantities of vitamin D, hypercalcemia and persistent biochemical hyperparathyroidism developed, and a parathyroid adenoma was removed. A history of nephrolithiasis and hypercalcemia preceding the institution of drug therapy allowed this patient's underlying parathyroid disease to be defined as primary hyperparathyroidism, which had been obscured by anticonvulsant therapy.

Aging and the neuroendocrine regulation of reproduction and body weight.

Aging in men is associated with a decline in trophic factors such as testosterone (T), alterations in body composition and impaired energy and body weight regulation. We performed studies to investigate the mechanisms underlying age-related changes in the neuroendocrine control of testis function, body composition, food intake and body weight in the Brown Norway (BN) rat. We found that similar to aging men, male BN rats demonstrate both primary and secondary testicular failure with aging without confounding age-related tumors, hormonal changes and systemic illnesses. With aging, these animals have blunted circadian variations in luteinizing hormone (LH) and T, and decreased hypothalamic gonadotropin-releasing hormone (GnRH) synthetic capacity with preserved pituitary gonadotropin responses to GnRH. We found that aging male BN rats have increased peripheral and visceral adiposity associated with increased insulin and leptin levels, and decreased relative lean body mass and muscle mass. We found that these rats exhibit reduced food intake and body weight gain associated with decreased hypothalamic neuropeptide Y (NPY) gene expression in the arcuate nucleus (ARC), both during ad-libitum feeding and after a 72-h fast. Recently, we found that old male BN rats treated chronically with troglitazone, an insulin sensitizer, lowered high insulin and leptin levels, decreased body fat, and corrected the blunted food intake and body weight gain response to fasting without affecting basal ARC NPY gene expression. These findings suggested that hyperinsulinemia and/or hyperleptinemia associated with aging may contribute to the age-related impairment in energy and weight regulation. Our studies suggest that the aging male BN rat is an excellent model to investigate the mechanisms underlying the age-associated changes in the neuroendocrine control of body composition, energy intake and body weight.

Economic outcome under Medicare prospective payment at a tertiary-care institution: the effects of demographic, clinical, and logistic factors on duration of hospital stay and part A charges for medical back problems (DRG 243).

We investigated the effects of prospectively identified factors on the duration of hospital stay and part A charges in 240 hospitalizations (of 230 patients) for the diagnosis-related group "medical back problems" (DRG 243) at a tertiary-care institution in 1985 to determine whether heterogeneity existed within this reimbursement category. We confirmed our initial postulates that nonosteoporotic fractures and neck problems, as well as hospitalizations primarily for myelography after outpatient neurologic evaluation, had considerably different economic outcomes and thus excluded these categories from further analysis. Statistical analysis (forward stepwise regression) of the remaining 132 patients who had "general medical back problems" showed that increasing age, associated osteoporosis, and therapeutic injections best explained variation in the natural logarithm of duration of stay (R2 = 0.16). Total number of diagnoses, spondylosis, associated osteoporosis, age, therapeutic injections, and performance of special procedures best explained the variation in the logarithm of part A charges (R2 = 0.29). The ability to identify factors within a specified category that affect the duration of hospitalization and part A charges jeopardizes the fairness of prospective payment, and we believe that DRG 243 should be adjusted for age, comorbidity, and readily identifiable clinical syndromes that have disparate economic consequences. Because of poorly substantiated efficacy and a significant association with longer hospital stays and higher part A charges, clinicians should review the use of therapeutic injections for medical back problems. Analysis of case-mix such as ours should be helpful in promoting efficient practice and ensuring the fairness of any reimbursement system.

Cross-sectional and longitudinal analysis of age-associated changes in body composition of male Brown Norway rats: association of serum leptin levels with peripheral adiposity.

Aging-associated alterations in body composition are accompanied by changes in the endocrine system. We evaluated, in male Brown Norway rats, the effects of aging on body composition and the association with serum levels of leptin, insulin, and testosterone. Body composition was assessed cross-sectionally in male rats (3, 8, 17, and 29 months) by a combination of dual energy x-ray absorptiometry (DEXA) and dissection of specific muscles and adipose depots. Longitudinal changes in body composition were quantified by DEXA before and after 3 months of ad-libitum feeding. Body weight, lean mass, absolute and percentage fat increased with age, whereas percentage of lean mass decreased. Leptin and insulin levels increased with age in proportion to adiposity; the increase in leptin with age was related to increased total and peripheral, but not visceral, fat. Testosterone decreased with age, and was associated with decreased lean and skeletal muscle mass. These findings suggest that alterations in body composition with age may be due to decreased trophic and increased lipogenic hormones. Relative to other rodent models, Brown Norway rats undergo shifts in body composition and in the hormonal milieu that are consistent with changes seen in aging humans.

Abnormal seminiferous tubule cells associated with choriocarcinoma: immunoperoxidase study.

A case is presented of widely metastatic choriocarinoma, associated with an intratubular proliferation of atypical germ cells in the testis and a minute adult cystic teratoma. Immunoperoxidase study of the intratubular germ cells did not confirm their trophoblastic origin. Intratubular proliferation of undifferentiated germ cells may be associated with choriocarcinoma.

Evaluation of a rat model of valproate-induced obesity.

Long-term treatment with the anticonvulsant valproate (VPA) leads to well-documented weight gain and obesity in humans. In an attempt to develop an animal model of this condition, adult rats were given VPA 20 g/kg (high-dose) or 2 g/kg (low-dose) in their daily feeding or orally 120 mg/kg body weight/day in two divided doses, and food intake and body weight were assessed. Valproate resulted in lower body weights in all protocols. Food intake was lower (p<0.001) for rats receiving high-dose VPA than for controls. Feed efficiency (change in weight divided by cumulative food intake for that period) was lower than that of controls for both high (p<0.0001) and low doses (NS). Metabolic rate and physical activity were not different between control and VPA animals, although decreased food intake would be expected to decrease metabolic rate. Valproate failed to produce obesity in rats in any treatment period. For reasons that are unclear, rats do not appear to be suitable as a model to study this adverse side effect of VPA in humans with epilepsy.

Interference in spectrophotometric analysis of cerebrospinal fluid by haemolysis induced by transport through a pneumatic tube system.

The hypothesis that sending blood-stained cerebrospinal fluid (CSF) through a pneumatic tube causes in vitro haemolysis has been tested. Spectrophotometric scanning of CSF supernatants demonstrated a significantly greater absorbance at 415 nm in those CSF samples that had been sent through the tube system compared to those that had not (P=0.0034). It is concluded that passage of blood-stained CSF down a pneumatic tube system causes in vitro haemolysis, accompanied by the release of oxyhaemoglobin from the lysed cells into the surrounding CSF. In view of this observation, it is recommended that CSF samples requiring spectrophotometric analysis, as part of the investigation of subarachnoid haemorrhage, should not be transported via a pneumatic tube system.

Comparison of neutrophil and monocyte function by microbicidal cell-kill assay in patients with cancer receiving granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or no cytokine after cytotoxic chemotherapy: a phase II trial.

Functional effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) were prospectively measured by harvesting blood samples from 51 oncology patients (21 who were receiving no cytokines, 14 receiving rhGM-CSF, and 16 who were receiving rhG-CSF) just before cytotoxic chemotherapy (baseline) immediately before the last cytokine dose (pre), 2 hours after the last cytokine dose (post), and 48 hours after the pre period (follow-up). Neutrophils and monocytes were separated and functional effects were measured by comparing cell-kill percentages, as determined by a microbial cell-kill assay against Staphylococcus aureus and Candida albicans. Optimal cell concentrations (2 x 10(6) monocytes/ml; 4 x 10(6) neutrophils/ml) and effector-to-cell ratios (1:50) were initially determined with blood samples harvested from 23 healthy volunteers. Results in oncology patients indicated that rhGM-CSF improved monocyte-killing activity against S. aureus at follow-up, compared with controls (p = 0.0094) and compared with monocytes from rhG-CSF-treated patients at the post period (p = 0.014). Cell-killing percentage of the rhGM-CSF-treated patients was also enhanced against C. albicans during the post period, compared with controls (p = 0.011) and rhG-CSF-treated patients (p = 0.067). Neutrophil activity was not altered by either cytokine. In conclusion, monocyte-induced microbial killing was enhanced in oncology patients receiving rhGM-CSF after cytotoxic chemotherapy, compared with patients receiving rhG-CSF or no cytokines. No differences in neutrophil activity were observed between patients receiving either cytokine.

Carotid duplex scan versus angiography in evaluation of carotid artery disease.

Angiography is presently felt to be the most accurate means of evaluating the carotid artery bifurcation, and is required preoperatively by most vascular surgeons. Arterial digital subtraction angiography (DSA) is the method commonly used. Angiography, however, is expensive and invasive, with significant morbidity and mortality. Duplex scanning (DS) has gained acceptance as a screening technique and has recently been advocated as the sole preoperative study in several centers because it is non-invasive and has an accuracy that is equal to or better than angiography. This study preoperatively evaluated 41 carotid arteries in 38 patients by arterial digital subtraction angiography and duplex scanning, and the results were compared to the surgical specimen. One artery was excluded because of an inadequate duplex scan, leaving 40 arteries studied by both techniques. Forty arteries (100%) studied by duplex scans corresponded with the surgical specimen, whereas only 31 arteries (77.5%) studied by DSA corresponded with the surgical specimen. Nine arteries (22.5%) studied by DSA had stenosis at the time of surgery significantly greater than predicted by DSA. Three arteries (7%) were felt to be normal by DSA, but had approximately 80 per cent stenosis by DS and at surgery. Our findings indicate that duplex scanning at our institution is more accurate than arterial DSA in determining percent diameter stenosis and plaque morphology of carotid arteries.

Pathology of surgically excised mitral valves. One hundred consecutive cases.

In this study, 100 consecutive, surgically excised, mitral valves were examined pathologically. The valves were classified according to primary conditions that resulted in valvular malfunction. Rheumatic mitral valvular diseases (stenosis and/or insufficiency) accounted for 54% of the cases. Myxomatous changes (prolapse) were present in 32 cases. Fifty-nine percent (19 cases) of those cases with myxomatous changes also had chordal rupture. Four of the cases had papillary muscle rupture, and in seven cases, papillary muscle dysfunction occurred. In one case bacterial endocarditis was observed on a previously normal valve. In one case the pathology of valvular. In one case the pathology of valvular changes was indeterminant. Lupus erythematosus was diagnosed in one patient, and mitral valve insufficiency may have resulted as a complication.