Rhodohalobacter mucosus sp. nov., isolated from a marine solar saltern.

A Gram-stain-negative, non-motile, aerobic, rod-shaped and reddish-pigmented bacterium, designated 8A47T, was isolated from a marine solar saltern located in Wendeng, PR China. The novel strain 8A47T grows at 20-42 °C, pH 7.0-9.0, and in the presence of 2.0-14.0% (w/v) NaCl. Optimal growth was observed at 37-40 °C, pH 7.5-8.0, and with 4.0-6.0% (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 8A47T formed an evolutionary lineage with members of the genus Rhodohalobacter. Strain 8A47T exhibited high level of similarity to Rhodohalobacter barlenses MCCC 1K03442T (94.7%) and Rhodohalobacter halophilus JZ3C29T (93.5%). The major fatty acids were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and iso-C15:0. The sole respiratory quinone was MK-7. The polar lipids of the new isolate consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified lipid, two unidentified phospholipids and three unidentified glycolipids and. The genomic DNA G + C content of the strain 8A47T was 47.7 mol%. Based on its phenotypic, chemotaxonomic, genotypic and genomic characteristics presented in this study, strain 8A47T is considered to represent a novel species of the genus Rhodohalobacter, for which the name Rhodohalobacter mucosus sp. nov. is proposed. The type strain is 8A47T (= KCTC 62603T = MCCC 1H00329T).

Impact of Herbal Preparations on Outcomes of Highly Active Antiretroviral Therapy: A One-Year Prospective Cohort Study.

OBJECTIVE: To investigate the impacts of two herbal preparations for human immunodeficiency virus/aquired immune deficiency syndrome (HIV/AIDS) patients, Shenling Fuzheng Capsule (, SLFZC) and Qingdu Capsule (, QDC), on the efficacy of highly active antiretroviral therapy (HAART). METHODS: HIV/AIDS patients met the criteria were all enrolled in a 1-year cohort study, in which patients receiving HAART alone were designated as Group A, those receiving HAART in combination with SLFZC were designated as Group B, and those receiving HAART in combination with QDC were designated as Group C, 100 cases in each group. The dose of SLFZC was 1.48 g (4 capsules), 3 times daily, and QDC 1.56 g (4 capsules), 3 times daily. T cell subsets, HIV RNA and HIV-1 drug resistance were detected at enrollment and 1 year after treatment. Patients were followed up every 3 months, during which side-effects and other clinical data were recorded. RESULTS: After 1-year treatment, the median increment in CD4 counts was 165.0, 178.0 and 145.0 cells/µL for Group A, B and C, respectively. HIV RNA was undetectable in 94% of patients in Group A, 96% in Group B and 92% in Group C. There were no differences regarding the increment in CD4 counts, HIV RNA and frequency of HIV-1 drug resistance mutations. Two of the 14 suspected side-effect symptoms, i.e. fatigue and dizziness, were lower in Groups B and C than in Group A (P<0.05, respectively) CONCLUSIONS: SLFZC and QDC do not have a negative impact on immunological and virological response to HAART; however, these preparations are not as potent in reducing HAART-associated side-effects as anticipated.

Halalkalibacillus sediminis sp. nov., isolated from sediment of sea cucumber culture pond.

A Gram-stain-positive, rod-shaped (0.3-0.4×1.2-2.0 µm), strictly aerobic and beige-pigmented bacterium, designated B3227T, was isolated from the sediment of a sea cucumber culture pond in Rongcheng, China (122.2° E 36.9° N). Its biochemical characteristics analysis revealed that the cells of this bacterium were catalase-positive and oxidase-negative. Cell growth occurred at 15-45 °C (optimum, 37 °C), pH 6.5-9.0 (pH 7.5-8.0) and in the presence of 0.0-22.0 % (w/v) NaCl (6.0-9.0 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain B3227T exhibited similarities of 95.7, 95.5, 95.5 and 95.3 % to the type strains of Filobacillus milensis, Piscibacillus salipiscarius, Halalkalibacillus halophilus and Piscibacillus halophilus, respectively, and the results of physiological analyses revealed that strain B3227T was most similar to the genus Halalkalibacillus. The cells were endospore-forming and comprised an A1-γ-meso-diaminopimelic acid-type peptidoglycan. The respiratory quinone of strain B3227T was MK-7, and the dominant fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. The genomic DNA G+C content was 38.7 mol%. The average nucleotide identity values between strain B3227T and H. halophilus JCM 14192T (ANIb 69.5%, ANIm 84.2 %) and F. milensis JCM 12288T (ANIb 70.1 %, ANIm 84.1 %) were below the cut-off level (95-96  %) for species delineation. The results of kegg analysis revealed that strain B3227T could biosynthesize shikimate acid, a base compound for the formulation of the swine flu drug. Based on its morphological and physiological properties, as well as phylogenetic distinctiveness, strain B3227T should be placed into the genus Halalkalibacillus as a representative of a new species, for which the name Halalkalibacillus sediminis sp. nov. is proposed. The type strain is B3227T (=KCTC 33093T=MCCC 1H00193T).

Chinese herbal medicine for patients living with HIV in Guangxi province, China: an analysis of two registries.

Anti-Retroviral Therapy (ART) is the recommended first line therapy for patients with HIV. Since 2004, Chinese government has provided free Chinese herbal medicine (CHM) for Chinese HIV/AIDS patients. Data of living patients with HIV from the NFTCMP database and Center for Disease Control (CDC) database during 2003-2016 in Guangxi province was obtained and compared. Patients were divided into 3 groups according to their recorded treatment regimens. A total of 2954 patients with their treatment recorded in the two databases were included for analysis, their median age was 46 years (IQR = 36-59), and 64.63% were male. CHM regimens users had baseline CD4 cell counts (380.11 ± 240.59 cell/µL), approximately 100 cell/µL significantly higher than patients receiving CHM combined with ART regimens or only ART regimens. There was no significant difference in mortality among groups. All three regimens improved patients' CD4 cell counts. Compared to the sharp improvement in ART group during the first 6 months, CD4 cell counts of patients in CHM group and CHM combined with ART group showed a smooth and steady rise. CD4 cell counts of the combined group remained much lower than ART group in the first 3 years, but overtook ART group in the fourth year.

Pontibacter arcticus sp. nov., isolated from rhizosphere soil of Saxifraga oppositifolia.

A Gram-stain-negative, rod-shaped, red-coloured and aerobic bacterium, designated 2b14T, was isolated from rhizosphere soil of Saxifraga oppositifolia sampled at the Chinese Arctic Yellow River Station in Norway. Optimal growth occurred at 28 °C (range, 4-37 °C) and pH 7.0-7.5 (pH 6.5-8.5). The strain could tolerate up to 2.5 % (w/v) NaCl concentration. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 2b14Thad the highest similarity value of 96.0 % to Pontibacter diazotrophicus CCTCC AB 2013049T. The major fatty acids were summed feature 4 (anteiso-C17 : 1 B and/or iso-C17 : 1 I), iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipid was found to be phosphatidylethanolamine. The genomic DNA G+C content of strain 2b14T was 45.5 mol%. The sole respiratory quinone was MK-7. The combined results of physiological, genotypic, phylogenetic and chemotaxonomic analyses showed that strain 2b14T represents a novel species within the genus Pontibacter, for which the name Pontibacter arcticus sp. nov. is proposed. The type strain is 2b14T (=KCTC 62596T=MCCC 1H00304T).

Rhodosalinus halophilus sp. nov., isolated from a marine solar saltern, and emended description of the genus Rhodosalinus.

A novel Gram-stain-negative, rod-shaped, beige-coloured, motile, facultatively anaerobic bacterium, designated as E84T, was isolated from sediment sampled from a marine solar saltern in Wendeng, PR China. Phylogenetic analysis based on 16S rRNA gene sequencing showed that Rhodosalinus sediminis WDN1C137T was the closest phylogenetic relationship, with 16S rRNA gene sequence similarity of 96.9 %. Optimal growth occurred at 33-37 °C (range, 20-40 °C), at pH 7.5 (pH 7.0-8.5) and with 6.0 % (w/v) NaCl (0.5-20.0 %). The sole respiratory quinone was Q-10. The major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C18 : 0 and cyclo C19 : 0ω8c. The polar lipids were phosphatidylglycerol, one unidentified glycolipid, two unidentified phospholipids and two unidentified lipids. The genomic DNA G+C content of strain E84T was 69.8 mol%. Based on the results of physiological, genotypic, phylogenetic and chemotaxonomic analyses, we concluded that strain E84T represents a novel species of the genus Rhodosalinus, for which the name Rhodosalinus halophilus sp. nov. is proposed. The type strain is E84T (=KCTC 52697T=MCCC 1H00231T).

Pedobacter changchengzhani sp. nov., isolated from soil of Antarctica.

A Gram-negative, aerobic, non-motile, pink and rod-shaped bacterium, designated E01020T, was isolated from soil collected from the Chinese Great Wall Station, Antarctica. Comparative analysis of 16S rRNA gene sequences revealed that strain E01020T is a member of the genus Pedobacter, related to Pedobacter alluvionis DSM 19624T (96.8% similarity), Pedobacter agri JCM 15120T (96.5% similarity) and Pedobacter chinensis JDX94T (96.3% similarity). The dDDH values and ANI values of strain E01020T with closely related strains indicate that it can be distinguished from them as a novel species. The DNA G+C content was determined to be 35.2 mol%. The growth of strain E01020T was observed at 4-25 °C (optimal 20 °C), in the presence of 0-1% NaCl (w/v, optimal 0%) and at pH 6.0-8.0 (optimal pH 7.0). Strain E01020T was found to contained menaquinon-7 (MK-7) as only respiratory quinone, iso-C15:0, iso-C17:0 3-OH and Summed feature 3 (C16:1ω6c and/or C16:1ω7c) as major fatty acids. The major polar lipids were phosphatidylethanolamine, one unidentified lipid and two unidentified aminolipids. On the basis of the results of the phylogenetic and phenotypic analyses, it was suggested that strain E01020T represents a novel species of the genus Pedobacter, for which the name Pedobacter changchengzhani sp. nov. is proposed. The type strain is E01020T (= KCTC 62990T = MCCC 1H00357T).

[Features of Syndrome Patterns of Chinese Medicine in Elderly AIDS Patients in Guangxi Zhuang Autonomous Region].

Objective To observe the features of syndrome patterns of Chinese medicine (CM) in elderly human immunodeficiency virus/acquired immune deficiency syndrome ( HIV/AIDS) patients in Guangxi Zhuang Autonomous Region. Methods According to a case-control study, a clinical question- naire was designated in elderly HIV/AIDS patients older than 50 years and healthy examinees with age and sex match. Their syndrome information of CM were collected from designated medical institutions in Guangxi Zhuang Autonomous Region from October 2013 to April 2014. Analyses of syndrome factors were conducted using WF-I[A Diagnosis and Treatment System of Traditional Chinese Medicine (Auxilia- ry). The disease location of CM and nature of diseases were compared between elderly HIV/AIDS patients and the controls. The features of syndrome patterns of CM in elderly HIV/AIDS patients were summarized. Results A total of 417 elderly HIV/AIDS patients and 362 examinees were enrolled. In elderly patients with HIV/AIDS, established syndrome factors of disease nature were qi deficiency, yang deficiency, yin deficiency, blood deficiency, dampness, and phlegm , and established syndrome factors of disease loca- tion included Shen, Fei, Pi, and Gan. There were statistical differences in established syndrome factors of disease location or nature between elderly patients with HIV/AIDS and the controls (P <0. 05). Conclu- sions Elderly HIV/AIDS patients were characterized by deficiency of qi, yang, yin, and blood in Shen, Fei, Pi, and Gan, as well as endogenous production of pathogenic factors such as dampness and phlegm. Intermingled deficiency and excess was dominated in elderly HIV/AIDS patients, and mainly man- ifested as deficiency syndrome.

[Thread-moxa in Zhuang folk medicine combined with acupuncture and external application drugs on AIDS patients with herpes zoster: a clinical observation].

OBJECTIVE: To observe the efficacy of thread-moxa in Zhuang folk medicine (TM) combined with acupuncture and external application drugs for AIDS patients with herpes zoster (AHZ). METHODS: A randomized, controlled clinical trial was conducted in 60 patients with AHZ. They were randomly assigned to the treatment group (treated with TM combined with acupuncture and Jingwanhong Scald Ointment) and the control group (treated with Famciclovir Tablet, nimesulide dispersible tablet, vitamin B1, ribavirin ointment). The treatment course was 14 days for both groups.The clinical efficacy, significant efficiency visual analog scale score (VAS), sleep quality score (QS), the postherpetic neuralgia rate in 1 year after treatment were observed. RESULTS: The markedly effective rate was significantly higher in the treatment group than in the control group (86.7% vs. 53.3%, P < 0.01). There was no statistical difference in the total effective rate between the two groups (96.7% vs. 80.0%, P > 0.05). The post-treatment VAS, QS, the time for pain disappearance, skin repair, crusting, and 1-year postherpetic neuralgia incidence rate were significantly lower in the treatment group than in the control group (P < 0. 05, P < 0.01). CONCLUSIONS: TM combined with acupuncture and Jingwanhong Scald Ointment was effective for treating AHZ patients. It relieved pain quickly, shortened their course of disease, and improved their quality of sleep.

Treatment of posthepatitic cirrhosis by Fuzheng Huayu Tablet for reinforcing qi and resolving stasis.

OBJECTIVE: To investigate the efficacy and safety of the Fuzheng Huayu Tablet FZHYT), which is used to reinforce qi and resolve stasis in patients with posthepatitic cirrhosis (PHC). METHODS: A multicenter, randomized, controlled clinical trial was conducted in 180 patients with PHC. The patients were randomly assigned using random numbers to a treatment group treated with FZHYT and a placebo group; the treatment course was 6 months for both groups. Overall response, adverse events (AEs), and the 2-year survival rate were assessed after treatment. Evaluations were made on changes in liver function, liver fibrosis, coagulation, hemodynamics, degrees of esophagogastric varices, ascites, quality of life (QOL), and scores of main symptoms. RESULTS: The overall response was significantly higher in the treatment group than the placebo group (86.7% vs. 62.2%, P<0.01). Patients in both groups had significant improvements in liver function [total bilirubin (TBIL), albumin (ALB)], liver fibrosis [hyaluronic acid (HA), type IV collagen (CIV)], coagulation [prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and thrombin time (TT)], hemodynamics portal venous flow (PVF), and splenic vein flow (SVF) after treatment. Between-group comparisons showed that compared with the placebo group patients in the treatment group achieved significantly greater improvements in TBIL, ALB, HA, C IV, PT, APTT, PVF, SVF, time to ascites resolution, 2-year survival, QOL, and symptom scores (P<0.05 or P<0.01). There were no significant AEs during the treatment. CONCLUSION: FZHYT is effective and safe for the treatment of hepatic cirrhosis as it is associated with improved liver function, liver fibrosis, coagulation, portal hypertension state, QOL, 2-year survival rate, and fewer AEs.

[Treating HIV/AIDS patients by shenlin fuzheng capsule and qingdu capsule: an analysis of clinical effectiveness].

OBJECTIVE: To analyze the clinical effectiveness of Shenling Fuzheng Capsule (SFC) and Qingdu Capsule (QC) in treating HIV/AIDS patients. METHODS: Totally 220 patients with complete clinical data, who received consecutive treatment for 6 months were selected from the database. They were assigned to two groups whether they would rather receive antiretroviral drugs, the Chinese medicine (CM) treatment group and the integrative medicine (IM) group. The 129 patients in the CM group were treated with SFC or QC, while the 91 patients in the IM group were treated with SFC or QC combined highly active antiretroviral agents. Total score and single score of clinical symptoms and signs, Karnofsky Performance Status (KPS), and changes of body weight before treatment, 3 and 6 months after treatment were compared. CD4+ cell counts were compared between before treatment and 6 months after treatment. RESULTS: The total score of clinical symptoms and signs were lower at 3 and 6 months of treatment than before treatment respectively (P < 0.01). The single score of clinical symptoms and signs such as cough, weakness, shortness of breath, vomit, spontaneous perspiration, hair loss,and chest pain were also lowered at 3 and 6 months of treatment (P < 0.05, P < 0.01), and the KPS increased (P < 0.05). The body weight increased (P < 0.05) and CD4 cell counts decreased (P < 0.05) in the CM group. There was no statistical difference in body weight or CD4 cell counts in the IM group between before and after treatment. CONCLUSION: SFC and QC could improve clinical symptoms and signs of HIV/ AIDS patients, but failed to deter the decrease of CD4+ cell counts.

A clinical study of weining granules in the treatment of gastric precancerous lesions.

OBJECTIVE: To investigate the clinical effects of Weining granules on gastric precancerous lesions (GPLs). METHODS: 120 patients with GPLs were randomly assigned in a 1:1 ratio to receive Weining granules (trial group) or the comparator Weifuchun tablets (control group) for 6 months. Outcomes were compared between the two groups including: overall response; gastroscopically-determined response; pathologically-confirmed response; eradication of Helicobacter pylori (HP); microvessel density (MVD) in the gastric mucosa; expression of vascular endothelial growth factor (VEGF); interleukin 2 (IL-2); interleukin 6 (IL-6); T lymphocyte subsets; immunoglobulins; symptom scores; quality of life (QOL); and adverse reactions. RESULTS: Patients in the trial group had a significantly higher (P < 0.05) overall response rate (81.7%) as compared with those in the control group (63.3%). Relative to treatment with Weifuchun tablets treatment with Weining granules resulted in a significant improvement (P < 0.05) in the scores for gastric pain, distension and stuffiness in the hypochondrium, and anorexia. As compared with the tablets the granules were associated with a significantly higher overall gastroscopically-determined response rate (78.3%; P < 0.05). Pathological examination of tissue samples indicated that 61.7% of patients receiving the granules were cured with an overall response rate of 75.5%; these rates were significantly higher than in the control group (P < 0.05). In comparison with patients receiving the tablets, those given the granules were significantly more likely to have their HP eradicated (75.0% vs. 51.4%; P < 0.05). Improvements in MVD, VEGF, CD4+, CD4+/ CD8+, IL-2, IL-6 and IgG were significantly greater with the Weining Granules than with the Weifuchun tablets (P < 0.05 or P < 0.01). After follow-up of 1 year, 17.5% of patients in the trial group relapsed as compared with 39.5% in the control group (P < 0.05). Relative to the control group, the trial group showed significantly greater improvements in physical, psychological and social relationships, and in environmental domains (P < 0.05 or P < 0.01). No significant adverse reactions were observed during treatment. CONCLUSION: The Weining granules appear to be effective in improving the gastric precancerous state and the main symptoms, in inhibiting angiogenesis, enhancing immune function and QOL, and in reducing 1-year relapses. In addition, this preparation seems to be associated with a low risk of adverse events, making it a safe and efficacious option for the treatment of GPLs.

[Randomized controlled trial on effect of Tuina for treatment of sub-health people of somatic pain].

OBJECTIVE: To evaluate the clinical effect and safety of Tuina for treatment of somatic pain of sub-health. METHODS: A randomized, double-blind and blank parallel controlled trial was done. The experiment group was treated with Tuina and the control group lied down for rest, 45 minutes each time, twice each week for three weeks. RESULTS: Tuina treatment could improve more on sensory, affective, evaluation, pain rating index and extant pain intensity of the pain index, and score of subjective sensation of life quality and health status together with physiology and psychology field of life quality. CONCLUSION: Massage is an effective therapy for treatment of somatic pain of sub-health without adverse reactions and it should be generalized to application.

[The combined effect of isoniazid and rifampicin on the activities of CYP4501A2 and CYP4503A4 in primary hepatocytes from healthy human adults].

OBJECTIVE: To study the combined effect of isoniazid and rifampicin on the activities of CYP1A2 and 3A4 in primary hepatocytes from healthy human adults. METHODS: The primary hepatocytes were isolated from adult healthy human livers, and cultured for 3 days. Then the cells were divided into 15 groups including two negative control groups (culture media alone) and 13 drug intervention groups, to which the following drugs were added: isoniazid (25 micromol/L, 50 micromol/L), rifampicin (12.5 micromol/L, 25 micromol/L) or both of them with different concentrations (CYP 1A2: rifampicin 12.5 micromol/L + isoniazid 50 micromol/L, rifampicin 25 micromol/L + isoniazid 50 micromol/L; CYP3A4: rifampicin 12.5 micromol/L + isoniazid 50 micromol/L, rifampicin 25 micromol/L + isoniazid 25 micromol/L, rifampicin 25 micromol/L + isoniazid 50 micromol/L) respectively. All the concentrations were consistent with the range of maximum clinical blood concentrations. After culture for 2 days, substrates (phenacetin for CYP1A2 , testosterone for CYP3A4) were added, and then the peak area (unit: mAU. min) of their metabolites was measured with high performance liquid chromatography (HPLC) to assess the activities of CYP450 1A2 and 3A4. RESULTS: (1) The activity of CYP1A2 in isoniazid groups with concentrations of 25 micromol/L and 50 micromol/L was (3.33 +/- 0.65), (3.03 +/- 0.38) mAU.min respectively, significantly different compared with that in the negative control group [(5.23 +/- 0.31) mAU.min, P < 0.01]. The activity of CYP450 1A2 in rifampicin groups with a concentration of 12.5 micromol/L was (6.07 +/- 0.55) mAU.min, which had significant difference compared with that in the negative control group (P < 0.05). There was no statistical difference of CYP1A2 activity between rifampicin with 25 micromol/L [(4.93 +/- 0.57) mAU.min] and the negative control group (P > 0.05). The activity of CYP1A2 of groups with two kinds of different concentrations of isoniazid and rifampicin combined groups was (3.27 +/- 0.96), (3.97 +/- 0.25) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.05). (2) The activity of CYP3A4 in isoniazid groups with concentrations of 25 micromol/L and 50 micromol/L was (5.40 +/- 1.35), (2.63 +/- 0.06) mAU.min respectively, which had significant difference compared with that in the negative control group [(12.53 +/- 0.51) mAU.min, P < 0.01]. The activity of CYP3A4 in rifampicin groups with concentrations of 12.5 micromol/L and 25 micromol/L was (165.17 +/- 11.47), (120.20 +/- 15.73) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.01). The activity of CYP3A4 in the three isoniazid and rifampicin combined groups with three kinds of different concentrations was (118.37 +/- 8.90), (77.53 +/- 6.91), (68.73 +/- 4.72) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.01), but they were lower than those in rifampicin groups with corresponding concentrations (P < 0.05). CONCLUSIONS: Isoniazid and rifampicin in the range of maximum clinical blood concentration have no significant inducing or inhibiting effect on the activity of CYP1A2 of healthy adult human primary hepatocytes. Isoniazid in the range of maximum clinical blood concentration can inhibit the activity of CYP3A4, while rifampicin can induce the activity of CYP3A4; the combined effect of isoniazid and rifampicin being the induction of CYP3A4 activity, but the inducing effect was less than that of rifampicin alone with the same concentration.

Conformational changes involved in MscL channel gating measured using FRET spectroscopy.

We demonstrate that fluorescence resonance energy transfer spectroscopy is a powerful tool for in situ structural analysis of multimeric membrane proteins by measuring the conformational changes involved in gating the mechanosensitive ion channel of large conductance. Ensemble analysis is used to analyze the intensity of light emitted by AlexaFluor-labeled cysteine mutants reconstituted into artificial liposomes before and after acceptor photobleaching. The diameter of the protein is found to increase by 16 A upon channel activation.

The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity.

The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively. We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (I24C-SL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin-spin interaction with the neighboring I24C-SL residues in the MscL channel pentamer. The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Interleukin-1beta enhances NMDA receptor-mediated current but inhibits excitatory synaptic transmission.

Interleukin (IL)-1beta is often characterized as the prototypic proinflammatory cytokine but is involved in various pathophysiological conditions in the central nervous system (CNS). A whole-cell recording technique was used to observe its effect on N-methyl-D-aspartate (NMDA)-evoked currents and spontaneous synaptic activity in cultured rat hippocampal neurons. The results showed that the frequencies but not the amplitudes of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC) were decreased by 10 or 100 ng/ml IL-1beta. IL-1beta at these concentrations also increased the NMDA receptor-mediated current. In addition, 10 ng/ml IL-1beta significantly increased the amplitude of the voltage-dependent Ca2+ current (I(Ca)). The increase in I(Ca) following treatment of cultures with IL-1beta resulted mainly from an increase in L-type current. These data suggest that IL-1beta modulates hippocampus-related functions via its effect on synaptic activity and Ca2+ signaling in neurons.