Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca²⁺-activated K⁺ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit.     

Association between CD14 Promoter -159C/T Polymorphism and the Risk of Sepsis and Mortality: A Systematic Review and Meta-Analysis

Background

Recent studies on the association between CD14-159C/T polymorphism and sepsis showed inconclusive results. Accordingly, we conducted a comprehensive literature search and a meta-analysis to determine whether the CD14-159C/T polymorphism conferred susceptibility to sepsis or was associated with increased risk of death from sepsis.

Methodology

Data were collected from the following electronic databases: PubMed, Embase, Medline, Web of Knowledge, and HuGE Navigator, with the last report up to June 15, 2012. The odds ratio (OR) and 95% confidence interval (CI) were used to assess the strength of association. We summarized the data on the association between CD14-159C/T polymorphism and sepsis in the overall population and subgroup by ethnicity and sepsis subtype.

Principal Findings

A total of 16 studies on sepsis morbidity (1369 cases and 2382 controls) and 4 studies on sepsis mortality (731 sepsis patients) met the inclusion criteria for meta-analysis. Overall analysis showed no strong evidences of association with sepsis susceptibility under any genetic model. However, slight associations were found in Asian populations (dominant model: OR = 1.38, 95%CI = 0.96–1.98, P = 0.08) and septic shock patients (dominant model: OR = 1.72, 95%CI 1.05–2.83, P = 0.03; allelic model: OR = 1.52, 95%CI 1.09–2.12, P = 0.01) in the stratified analysis. Moreover, there was borderline association between CD14-159C/T and sepsis mortality under the dominant genetic model (OR = 1.44, 95%CI = 0.98–2.11, P = 0.06).

Conclusions/Significance

This meta-analysis suggests that the CD14-159C/T polymorphism may not be a significant susceptibility factor in the risk of sepsis and mortality. Only weak associations were observed in Asian populations and septic shock patients. More studies based on larger sample sizes and homogeneous sepsis patients are needed to confirm these findings.     

PAI-1 -675 4G/5G Polymorphism in Association with Diabetes and Diabetic Complications Susceptibility: a Meta-Analysis Study

A meta-analysis was performed to assess the association between the PAI-1 -675 4G/5G polymorphism and susceptibility to diabetes mellitus (DM), diabetic nephropathy (DN), diabetic retinopathy (DR) and diabetic coronary artery disease (CAD). A literature-based search was conducted to identify all relevant studies. The fixed or random effect pooled measure was calculated mainly at the allele level to determine heterogeneity bias among studies. Further stratified analyses and sensitivity analyses were also performed. Publication bias was examined by the modified Begg’s and Egger’s test. Twenty published articles with twenty-seven outcomes were included in the meta-analysis: 6 studies with a total of 1,333 cases and 3,011 controls were analyzed for the PAI-1 -675 4G/5G polymorphism with diabetes risk, 7 studies with 1,060 cases and 1,139 controls for DN risk, 10 studies with 1,327 cases and 1,557 controls for DR and 4 studies with 610 cases and 1,042 controls for diabetic CAD risk respectively. Using allelic comparison (4G vs. 5G), the PAI-1 -675 4G/5G polymorphism was observed to have no significant association with diabetes (REM OR 1.07, 95% CI 0.96, 1.20), DN (REM OR 1.10, 95% CI 0.98, 1.25), DR (REM OR 1.09, 95% CI 0.97, 1.22) or diabetic CAD risk (REM OR 1.07, 95% CI 0.81, 1.42), and similar results were obtained in the dominant, recessive and co-dominant models. Our meta-analyses suggest that the PAI-1 -675 4G/5G polymorphism might not be a risk factor for DM, DN, DR or diabetic CAD risk in the populations investigated. This conclusion warrants confirmation by further studies.     

The Relationship between Polyamines and Hormones in the Regulation of Wheat Grain Filling

The grain weight of wheat is strongly influenced by filling. Polyamines (PA) are involved in regulating plant growth. However, the effects of PA on wheat grain filling and its mechanism of action are unclear. The objective of the present study was to investigate the relationship between PAs and hormones in the regulation of wheat grain filling. Three PAs, spermidine (Spd), spermine (Spm), and putrescine (Put), were exogenously applied, and the grain filling characteristics and changes in endogenous PA and hormones, i.e., indole-3-acetic acid (IAA), zeatin (Z) + zeatin riboside (ZR), abscisic acid (ABA), ethylene (ETH) and gibberellin 1+4 (GAs), were quantified during wheat grain filling. Exogenous applications of Spd and Spm significantly increased the grain filling rate and weight, but exogenous Put had no significant effects on these measures. Exogenous Spd and Spm significantly increased the endogenous Spd, Spm, Z+ZR, ABA, and IAA contents and significantly decreased ETH evolution in grains. The endogenous Spd, Spm and Z+ZR contents were positively and significantly correlated with the grain filling rate and weight of wheat, and the endogenous ETH evolution was negatively and significantly correlated with the wheat grain filling rate and weight. Based upon these results, we concluded that PAs were involved in the balance of hormones that regulated the grain filling of wheat.     

Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation

The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.     

Efficacy and Tolerability of the Fixed Combinations Latanoprost/Timolol versus Dorzolamide/Timolol in Patients with Elevated Intraocular Pressure: A Meta-Analysis of Randomized Controlled Trials

Objective

To evaluate the efficacy and tolerability of the fixed combination of Latanoprost/Timolol versus Dorzolamide/Timolol in the treatment of patients with elevated intraocular pressure (IOP).

Methods

A comprehensive literature meta-analysis was performed according to the Cochrane Collaboration methodology to identify randomized clinical trials comparing latanoprost/timolol FC (FCLT) with dorzolamide/timolol (FCDT) in patients with elevated IOP. The efficacy estimates were measured by the weight mean difference (WMD) for the IOP reduction (IOPR) from baseline to end point, including the diurnal mean IOPR, 8 AM IOPR, 12 PM IOPR, and 4 PM IOPR. The tolerability estimates were measured by RR for adverse events. All outcomes were reported with a 95% confidence interval (CI). The data were synthesized by Stata 12.0 SE for Windows.

Results

Eight studies involving 841 patients (841 eyes) were included in the meta-analysis. With a WMD of IOPR in the diurnal mean of 0.16 mmHg (95% CI, -0.31 to 0.63), the FCLT was as effective as FCDT in lowering IOP in patients with elevated IOP (P = 0.51). The WMDs of IOPR were 0.58 mmHg (95% CI: -0.002 to 1.17) at 8 AM, -0.07 mmHg (95% CI: -0.50 to 0.36) at 12 PM, and 0.41 mmHg (95% CI: -0.18 to 1.00) at 4 PM, and there were no significant difference between FCLT and FCDT. FCLT was associated with a significantly lower incidence of eye pain, bitter taste, and irritation/stinging than FCDT, with pooled RRs of 0.34 (95% CI: 0.14 to 0.82), 0.06 (95% CI:0.008 to 0.42), and 0.35 (95% CI: 0.14 to 0.85), respectively.

Conclusion

FCLT was associated with equivalent efficacy in IOP lowering comparing with FCDT. However, FCLT was better tolerated than FCDT.     

Hydrogen Peroxide Is a Second Messenger in the Salicylic Acid-Triggered Adventitious Rooting Process in Mung Bean Seedlings

In plants, salicylic acid (SA) is a signaling molecule that regulates disease resistance responses, such as systemic acquired resistance (SAR) and hypertensive response (HR). SA has been implicated as participating in various biotic and abiotic stresses. This study was conducted to investigate the role of SA in adventitious root formation (ARF) in mung bean (Phaseolus radiatus L) hypocotyl cuttings. We observed that hypocotyl treatment with SA could significantly promote the adventitious root formation, and its effects were dose and time dependent. Explants treated with SA displayed a 130% increase in adventitious root number compared with control seedlings. The role of SA in mung bean hypocotyl ARF as well as its interaction with hydrogen peroxide (H₂O₂) were also elucidated. Pretreatment of mung bean explants with N, N’-dimethylthiourea (DMTU), a scavenger for H₂O₂, resulted in a significant reduction of SA-induced ARF. Diphenyleneiodonium (DPI), a specific inhibitor of membrane-linked NADPH oxidase, also inhibited the effect of adventitious rooting triggered by SA treatment. The determination of the endogenous H₂O₂ level indicated that the seedlings treated with SA could induce H₂O₂ accumulation compared with the control treatment. Our results revealed a distinctive role of SA in the promotion of adventitious rooting via the process of H₂O₂ accumulation. This conclusion was further supported by antioxidant enzyme activity assays. Based on these results, we conclude that the accumulation of free H₂O₂ might be a downstream event in response to SA-triggered adventitious root formation in mung bean seedlings.     

Identification of Ochratoxin A Producing Fungi Associated with Fresh and Dry Liquorice

The presence of fungi on liquorice could contaminate the crop and result in elevated levels of mycotoxin. In this study, the mycobiota associated with fresh and dry liquorice was investigated in 3 producing regions of China. Potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxin B1 (AFB1) production using liquid chromatography/mass spectrometry/mass spectrometry. Based on a polyphasic approach using morphological characters, β-tubulin and RNA polymerase II second largest subunit gene phylogeny, a total of 9 genera consisting of 22 fungal species were identified, including two new Penicillium species (Penicillium glycyrrhizacola sp. nov. and Penicillium xingjiangense sp. nov.). The similarity of fungal communities associated with fresh and dry liquorice was low. Nineteen species belonging to 8 genera were detected from fresh liquorice with populations affiliated with P. glycyrrhizacola, P. chrysogenum and Aspergillus insuetus comprising the majority (78.74%, 33.33% and 47.06% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. In contrast, ten species belonging to 4 genera were detected from dry liquorice with populations affiliated with P. chrysogenum, P. crustosum and Aspergillus terreus comprising the majority (64.00%, 52.38% and 90.91% of total) of the community from Gansu, Ningxia and Xinjiang samples, respectively. Subsequent LC/MS/MS analysis indicated that 5 fungal species were able to synthesize OTA in vitro including P. chrysogenum, P. glycyrrhizacola, P. polonicum, Aspergillus ochraceus and A. westerdijkiae, the OTA concentration varied from 12.99 to 39.03 µg/kg. AFB1 was absent in all tested strains. These results demonstrate the presence of OTA producing fungi on fresh liquorice and suggest that these fungi could survive on dry liquorice after traditional sun drying. Penicillium chrysogenum derived from surrounding environments is likely to be a stable contributor to high OTA level in liquorice. The harvesting and processing procedure needs to be monitored in order to keep liquorice free of toxigenic fungi.