Functional analysis of CaIPT1, a sphingolipid biosynthetic gene involved in multidrug resistance and morphogenesis of Candida albicans

In the present study we describe the isolation and functional analysis of a sphingolipid biosynthetic gene, IPT1, of Candida albicans. The functional consequence of the disruption of both alleles of IPT1 was confirmed by mass analysis of its sphingolipid composition. The disruption of both alleles or a single allele of IPT1 did not lead to any change in growth phenotype or total sphingolipid, ergosterol, or phospholipid content of the mutant cells. The loss of mannosyl diinositol diphosphoceramide [M(IP)(2)C] in the ipt1 disruptant, however, resulted in increased sensitivity to drugs like 4-nitroquinoline oxide, terbinafine, o-phenanthroline, fluconazole, itraconazole, and ketoconazole. The increase in drug susceptibilities of ipt1 cells was linked to an altered sphingolipid composition, which appeared to be due to the impaired functionality of Cdr1p, a major drug efflux pump of C. albicans that belongs to the ATP binding cassette superfamily. Our confocal and Western blotting results demonstrated that surface localization of green fluorescent protein-tagged Cdr1p was affected in ipt1 disruptant cells. Poor surface localization of Cdr1p resulted in an impaired ability to efflux fluconazole and rhodamine 6G. The effect of mannosyl inositol phosphoceramide accumulation in the ipt1 mutant and the absence of M(IP)(2)C from the ipt1 mutant on the efflux of drug substrates was very selective. The efflux of methotrexate, a specific substrate of CaMdr1p, another major efflux pump of major facilitator superfamily, remained unaffected in ipt1 mutant cells. Interestingly, changes in sphingolipid composition affected the ability of mutant cells to form proper hyphae in various media. Taken together, our results demonstrate that an altered composition of sphingolipid, which is among the major constituents of membrane rafts, affects the drug susceptibilities and morphogenesis of C. albicans.     

Pretreatment with β‐adrenergic receptor agonists facilitates induction of LTP and sharp wave ripple complexes in rodent hippocampus

Norepinephrine, is involved in the enhancement of learning and memory formation by regulating synaptic mechanisms through its ability to activate pre‐ and post‐synaptic adrenergic receptors. Here we show that β‐agonists of norepinephrine facilitate the induction of both associational LTP and sharp wave ripples (SPW‐Rs) in acute slices of rat hippocampus in area CA3. Surprisingly, this facilitating effect persists when slices are only pretreated with β‐receptor agonists followed by wash out and application of the unspecific β‐adrenoreceptor (βAR) antagonist propranolol. During application of βAR agonists repeated stimulation resulted in facilitated induction of SPW‐Rs. Since SPW‐Rs are thought to be involved in memory replay we studied the effects of βAR‐agonists on spontaneous SPW‐Rs in murine hippocampus and found that amplitude and incidence of SPW‐Rs increased. These effects involve cyclic‐AMP and the activation of protein kinase A and suggest a supportive role in memory consolidation. © 2016 Wiley Periodicals, Inc.     

Implication of recent trials with b-hydroxy-b-methylglutaryl coenzyme A reductase inhibitors for hypertension management

BACKGROUND: There is broad agreement that statin treatment should be targeted at absolute coronary heart disease (CHD) risk but no consensus on the level of risk to target. We have examined the implications of adopting three different treatment policies for the management of hypertensive patients in the UK using data from treated hypertensives aged 35-69 years included in the Health Survey for England (1993). METHODS: We calculated the proportion of hypertensive patients with existing atherosclerotic cardiovascular disease requiring statin treatment for secondary prevention of CHD. For those without atherosclerotic cardiovascular disease (primary prevention), we estimated CHD risk from the Framingham equation and examined the proportion with CHD risk exceeding thresholds of 4.5, 3 and 1.5% per year. RESULTS: Twenty-one percent of treated hypertensives would require statin treatment for secondary prevention of CHD. When the CHD event threshold for statin treatment was set at > or =4.5% per year [equivalent to a number needed to treat (NNT) in 5 years of 13] a further 0.6% of hypertensive patients were identified for treatment; at a threshold of 3.0% per year (NNT = 20) 5.5% of patients were identified for primary prevention; and at a threshold of 1.5% per year (NNT = 40) 28.5% of patients were identified for primary prevention. CONCLUSIONS: Those needing secondary prevention are first priority for statins and 21% of hypertensive patients will require treatment Formulation of guidelines for primary prevention should take into account the NNT; the proportion of patients targeted for treatment; the cost-effectiveness and the total cost of treatment. Current British guidance will entail treating an additional 5.5% of hypertensive patients for primary prevention and therefore 27% of hypertensive patients.     

Evaluation of glutathione s‐transferase as toxicity indicator for roxarsone and arsanilic acid in Eisenia fetida

Different compounds can induce stress response by targeting specific genes. Studies related to elucidating the detoxification and adaptive responses of proteins like glutathione‐s‐transferase (GST) can be helpful in better understanding toxicity. Roxarsone and arsanilic acid, which have been exhaustively used as animal and poultry feed additives, pose a threat to the environment and human health. GST enzyme bioassay revealed fluctuations in response to different concentrations of roxarsone and arsanilic acid at different time intervals. The highest GST enzyme activity (40.51%) was observed on day 15 of treatment with roxarsone. On the other hand, arsanilic acid caused the maximum enzyme activity (52.11%) on day 10 of treatment. During this study, the full‐length gene sequence of GST, having the size 984 bp (Genbankno. HQ693699), was achieved from Eisenia fetida and established as a biomarker to assess the toxicity of roxarsone and arsanilic acid. The deduced protein has a computed molecular mass of 23.56 kDa and a predicted isoelectric point of 9.92. Quantitative real‐time PCR revealed significant differential gene expression in response to roxarsone and arsanilic acid treatment as compared with control treatment. Roxarsone caused the highest gene expression of 7.0‐fold increase over control on day 15 of treatment, whereas arsanilic acid resulted in the highest gene expression reaching to 14.56‐fold as compared with control. This study is helpful in understanding the role of GST as a potential biomarker for chemicals like roxarsone and arsanilic acid, which can pollute the food chain. Copyright © 2012 John Wiley & Sons, Ltd.     

Molecular characterization of Tobacco leaf curl Pusa virus, a new monopartite Begomovirus associated with tobacco leaf curl disease in India

Leaf curl disease of tobacco (TbLCD) is endemic in India. A monopartite Begomovirus, a betasatellite and an alphasatellite were found associated with the disease in Pusa, Bihar. The DNA-A of the Begomovirus associated with TbLCD in Pusa, Bihar was found to comprise of 2707 nt with a typical Old World begomovirus-like genome organization. The full-length sequence of DNA-A [HQ180391] showed that the Pusa isolate is a newly described member of the genus Begomovirus, as it had <89% sequence homology with DNA-A of all the known begomoviruses. The isolate is tentatively named as Tobacco leaf curl Pusa virus [India:Pusa:2010]. The betasatellite (HQ180395) associated with TbLCD in Pusa was identified as a variant of Tomato leaf curl Bangladesh betasatellite [IN:Raj:03], with which it shared 90.4% sequence identity. The alphasatellite (HQ180392) associated with the disease had highest 87% nucleotide sequence identity with Tomato leaf curl alphasatellite. The Begomovirus, betasatellite, and alphasatellite associated with TbLCD in Pusa, Bihar, India were found to be recombinants of extant begomoviruses, betasatellites and alphasatellites spreading in the Indian sub-continent and South-East Asia.     

Partial disinhibition is required for transition of stimulus-induced sharp wave-ripple complexes into recurrent epileptiform discharges in rat hippocampal slices

Sharp wave-ripple complexes (SPW-Rs) in the intact rodent hippocampus are characterized by slow field potential transients superimposed by close to 200-Hz ripple oscillations. Similar events have been recorded in hippocampal slices where SPW-Rs occur spontaneously or can be induced by repeated application of high-frequency stimulation, a standard protocol for induction of long-lasting long-term potentiation. Such stimulation is reminiscent of protocols used to induce kindling epilepsy and ripple oscillations may be predictive of the epileptogenic zone in temporal lobe epilepsy. In the present study, we investigated the relation between recurrent epileptiform discharges (REDs) and SPW-Rs by studying effects of partial removal of inhibition. In particular, we compared the effects of nicotine, low-dose bicuculline methiodide (BMI), and elevated extracellular potassium concentration ([K(+)](o)) on induced SPW-Rs. We show that nicotine dose-dependently transformed SPW-Rs into REDs. This transition was associated with reduced inhibitory conductance in CA3 pyramidal cells. Similar results were obtained from slices where the GABAergic conductance was reduced by application of low concentrations of BMI (1-2 μM). In contrast, sharp waves were diminished by phenobarbital. Elevating [K(+)](o) from 3 to 8.5 mM did not transform SPW-Rs into REDs but significantly increased their incidence and amplitude. Under these conditions, the equilibrium potential for inhibition was shifted in depolarizing direction, whereas inhibitory conductance was significantly increased. Interestingly, the propensity of elevated [K(+)](o) to induce seizure-like events was reduced in slices where SPW-Rs had been induced. In conclusion, recruitment of inhibitory cells during SPW-Rs may serve as a mechanism by which hyperexcitation and eventually seizure generation might be prevented.     

Glutathione as an antioxidant in inorganic mercury induced nephrotoxicity

Heavy metal toxicity represents an uncommon but clinically significant medical condition, which if unrecognized or inappropriately treated results in significant morbidity and mortality. Among heavy metals, mercury is recognized as a potent and widely distributed toxicant having the ability to accumulate at various levels of food chain besides possessing ability to cross placental and blood-brain barrier. Symptom picture of mercury (Hg 2+ ) toxicity is characterized mainly by a series of renal disorders. Mechanism of inorganic mercury toxicity includes production of reactive oxygen species (ROS) capable of damaging lipids in membrane, proteins or enzymes in tissues, and DNA to induce oxidative stress as balance between generation, and elimination of ROS is essential for maintaining the functional integrity of a cell. Mitigation of endogenous mercury depends as a part on the presence of antioxidants such as glutathione - most abundant intracellular non-protein thiol that plays a central role in the maintenance of cellular redox status by quenching free radicals generated during oxidative stress. Ability of a cell to survive the threat posed by endogenous mercury represents a biological adaptation fundamental to survival. This review describes the current understanding and the mechanisms involved by different forms of mercury in eliciting their toxicity in kidney along with the knowledge of major intracellular reductant that plays important role in the mitigation of mercury toxicity for the maintenance of homeostasis within the body of living organisms.     

Monoamines block kainate‐ and carbachol‐induced γ‐oscillations but augment stimulus‐induced γ‐oscillations in rat hippocampus in vitro

Monoamines are implicated in a cognitive processes in a variety of brain regions, including the hippocampal formation, where storage and retrieval of information are facilitated by synchronous network activities. We have investigated the effects of norepinephrine, serotonin, and dopamine on carbachol‐, kainate‐, and stimulus‐induced hippocampal γ‐oscillations employing combined extra‐ and intracellular recordings. Monoamines dose‐dependently and reversibly suppressed kainate‐ and carbachol‐induced γ‐oscillations while increasing the frequency. The effect of serotonin was mimicked by fenfluramine, which releases serotonin from presynaptic terminals. Forskolin also suppressed kainate‐ and carbachol‐induced γ‐oscillations. This effect was mimicked by 8‐Br‐cAMP and isoproterenol, an agonist of noradrenergic β‐receptor suggesting that the monoamines‐mediated suppression of these oscillations could involve intracellular cyclic adenosine 3′,5′‐cyclic monophosphate (AMP). By contrast, stimulus‐induced γ‐oscillations were dose‐dependently augmented in power and duration after monoamines application. Intracellular recordings from pyramidal cells revealed that monoamines prolonged the stimulus‐induced depolarization and membrane potential oscillations. Stimulus‐induced γ‐oscillations were also suppressed by isoproterenol, the D1 agonist SKF‐38393 forskolin, and 8‐Br‐cAMP. This suggests that the augmentation of stimulus‐induced γ‐oscillations by monoamines involves—at least in part—different classes of cells than in case of carbachol‐ and kainate‐induced γ‐oscillations. © 2009 Wiley‐Liss, Inc.     

131I activity for remnant ablation in patients with differentiated thyroid cancer: A systematic review

CONTEXT: Radioiodine ablation of the thyroid remnant after thyroidectomy is commonly performed in the management of patients with differentiated thyroid cancer. Although many centers administer an activity of 100 mCi, there is uncertainty over using a lower activity. OBJECTIVE: A systematic review of the published literature was used to compare the success rates of remnant ablation using approximately 30 mCi with approximately 100 mCi (1.1 vs. 3.7 GBq). DATA SOURCES: Data were obtained from MEDLINE and EMBASE for the years 1966 to March 2006. STUDY SELECTION: All studies that reported rates of successful ablation associated with approximately 30 or approximately 100 mCi of radioiodine were reviewed. DATA EXTRACTION: Studies were based on reviews of patient case notes (n = 41), prospective cohorts (n = 12), and randomized trials (n = 6). We obtained the success of thyroid remnant ablation according to different administered activities of radioiodine. Where a study reported on two or more activities, the risk ratio of having a successful ablation (approximately 30 vs. approximately 100 mCi) was calculated and combined in a meta-analysis. DATA SYNTHESIS: Observational studies confirmed the high ablation success rate ( approximately 80%) using approximately 100 mCi, although 22% of studies reported a rate of 90% or greater. The pooled ablation success rate in these studies was 10% lower using 30 mCi compared with 100 mCi (95% confidence interval, 3-17%; P = 0.01). The meta-analysis of the randomized trials produced equivocal results. For example, the rate of successful ablation in patients given 30 mCi was 8% lower compared with 100 mCi (95% confidence interval, 29% lower or up to 20% greater, P = 0.58), consistent with there being no difference or that 30 mCi is much less effective. CONCLUSIONS: From the published data, it is not possible to reliably determine whether ablation success rates using 30 mCi are similar to using 100 mCi. Large randomized trials are needed to resolve the issue and guide clinical practice.     

Adjuvant high-dose-rate and low-dose-rate brachytherapy with external beam radiation in soft tissue sarcoma: a comparison of outcomes

PURPOSE: To compare outcomes of patients treated with low-dose-rate (LDR) adjuvant brachytherapy (BT) + external beam radiation therapy (EBRT) vs. high-dose-rate (HDR) adjuvant BT + EBRT. METHODS AND MATERIALS: Thirty-seven patients with soft tissue sarcoma (STS) were treated with pre- or postoperative external beam irradiation and postoperative LDR or HDR BT. RESULTS: There was no significant difference in the 2-year local control rates (90% with LDR boost vs. 94% for HDR). The rate of National Cancer Institute (NCI) grades 2-4 wound-healing complications was 40% in the LDR group vs. 18% in the HDR group (p = 0.14). On univariate analysis, only suboptimal geometry of the implant predicted for increasing complication rate in the LDR group. In the HDR group, BT dose per fraction, total BT dose, and total biologically equivalent dose (BED3) radiation dose were significant. CONCLUSIONS: HDR and LDR boost BT results in acceptable local control for STS. There is a suggestion that HDR may have lower incidence of severe (grade > or = 3) acute toxicity than LDR.