A rapid, sensitive and convenient LC-MS/MS method was developed for the determination of γ-aminobutyric acid (GABA) in human plasma. d2-γ-Aminobutyric acid (d2-GABA) was synthesized as internal standard (IS). After extraction from human plasma by protein precipitation with acetonitrile, all analytes were separated on a Luna HILIC column (100 mm x 3.0 mm, 3 μm) using an isocratic mobile phase of water: acetonitrile: formic acid (20 : 80 : 0.12) with a flow rate of 0.5 mL x min(-1). Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode (MRM) in positive electrospray ionization using the transitions of m/z 104 --> 69 for GABA and m/z 106 --> 71 for d2-GABA. The method was linear in the concentration range of 5.00 to 1 000 ng x mL(-1). The intra- and inter-day precisions were within 9.9%, and accuracy ranged from 99.1% to 104%, within the acceptable limit across all concentrations. The method was successfully applied to a pharmacokinetic study of GABA tablets in healthy Chinese volunteers.
Chromatography, Liquid ; Humans ; Tandem Mass Spectrometry ; gamma-Aminobutyric Acid ; blood

Objective To investigate the effect of microRNA-101 (miRNA-101) on atrial fibrosis in human chronic atrial fibrillation (AF). Methods Right atrial appendages were obtained from 59 patients (30 with AF) undergoing cardiac surgery, including 47 patients with valve heart disease and 12 patients with congenital heart disease. The expression of miRNA-101 was determined by quantitative real-time PCR in the right atrial appendages of patients with and without AF. The cell-specific localization of miRNA-101 was detected by in situ hybridization assay. The mRNA and protein expression levels of transforming growth factor β typeⅠreceptor (TGFβRⅠ) and collagen type I (COL1) were determined by quantitative real-time PCR and Western-blot assay, respectively. Collagen in the right atrial appendages was observed by Masson staining assay. Results The expression of miRNA-101 was found to be significantly down-regulated in AF patients compared with patients with sinus rhythm (SR) (P < 0.05). The result of miRNA-ISH showed that miRNA-101, which was highly distributed within the connective tissues of heart, was down-regulated at about 24.9% in patients with AF compared with patients with SR. No significant differences at the mRNA expression level of TGFβRI was found between patients with AF and patients with SR (P > 0.05). But the protein expression of TGFβRI in patients with AF was significantly higher than that of patients with SR (P < 0.05). The mRNA and protein expressionsl of COL1 were significantly higher in patients with AF than thoset of patients with SR (P < 0.05). The collagen was significantly increased in patients with AF than that of patients with SR (P < 0.05). Conclusions Downregulation of miRNA-101 may contribute to atrial fibrosis in human atrial fibrillation by targeting TGFβRⅠ.

Objective To investigate the main genotypes and the epidemic characteristics of TEM-type and SHV-type extended-spectrum beta-lactamases (ESBLs) of the gram-negative bacilli in Guangzhou.Methods The phenotype of ESBLs from 3 500 clinical isolates were primary screened and confirmed by the NCCLS confirmatory test according to guidelines of NCCLS(2001). PCR were performed on 3 500 clinical isolates using primers specific for blaTEM and blaSHV, respectively, the PCR product were purified and sequenced by ABI prism 377 DNA sequencer.Results Total of 3 500 un-replicated and consecutive Gram-negative bacilli were isolated from 13 hospitals in Guangzhou in the past two years, and the prevalence of ESBLs-producing clinical Gram-negative isolates was 31.0%(1 084/3 500). The positive rates of PCR results for blaTEM, blaSHV, and both of them in all the clinical Gram-negative isolates were 24.0%(840/3 500),10.8%(378/3 500),and 3.7%(128/3 500), respectively. All PCR products for blaTEM were further identified as TEM-1-type non-ESBLs gene, including TEM-1,TEM-1B,TEM-1D,and TEM-1F,by DNA sequencing analyses. However, almost all of the blaSHV gene were further identified as SHV-type ESBLs gene(the prevalence of SHV-1 is 7.2% only).The prevalence of SHV-12/5a accounted for highest(50.0%,152/304) in Guangzhou. Our test also showed that 53%(340/641) of blaTEM-producers [JP2] were Escherichia coli, and 57.9%(176/304) of blaSHV-producers were Klebsiella pneumoniae.Conclusions [JP]We could conclude from above results that SHV-type ESBLs, especial SHV-12/5a, was the prevalent genotype of ESBLs of clinical gram-negative bacilli in Guangzhou. TEM-type ESBLs did not exist in our city. In addition, SHV-12/5a-producing strains probably were main epidemic strains in Guangzhou. As for detection of ESBLs with regular phenotype methods, there was possibility of false negative and false positive.

Depression is a common emotional disorder that seriously affects people's life and health all over the world. The pathogenesis of depression is complex, and traditional Chinese medicine (TCM) for antidepressants has a good therapeutic effect because of its multi-component, multi-pathway, and multi-target action mode. At present, the anti-depressive mechanism of TCM has not been fully clarified, but it is clear that depression is closely related to metabolic health. Therefore, in order to further explore the anti-depressive mechanism of TCM, this paper proposes research strategies on the anti-depressive mechanism of TCM based on functional metabolomics from the perspective of metabolism, the potential biomarkers of depression are analyzed with the help of multi-omics combined analysis technology, and the functional molecules of TCM for antidepressant are studied. Molecular biology techniques are used to accurately capture the molecular interactions between biomarkers of depression and functional compounds, which identify effective drug targets and further elucidate the biochemical functions and related mechanisms involved in depression metabolic disorders. This paper systematically reviews the research strategies and applications of functional metabolomics in the anti-depressive mechanisms of TCM, expounds on the core value of functional metabolomics, and summarizes the current research status and hot issues of TCM for antidepressants in recent years, providing new methods and new ideas for the study of mechanisms of TCM with the help of functional metabolomics.

Objective To evaluate the feasibility and clinical value of combined clonidine and sleep-deprivation induced seizures for ictal brain SPECT imaging in patients with epilepsy. Methods Fiftytwo epilepsy patients were given oral clonidine plus sleep-deprivation to induce seizures with video-electroencephalogram (VEEG) monitoring. Forty-seven patients were selected as control group, whose seizures were induced by sleep-deprivation only. 99Tcm-ethylcysteinate dimer (ECD) was injected within 30 s since a clinical sign and/or a typical EEG discharge of epilepsy was recognized. Brain SPECT was performed 30 min after 99TcmECD injection. X2-test was performed by using software SPSS 10. 0. Results One to two hrs after oral intake of clonidine plus sleep-deprivation, 75% (39/52) patients were induced seizures, including 92.3% (36/39) with subclinical seizures and 7.7% (3/39) with clinical seizures. Ictal brain SPECT localized the lesions with high uptake of 99Tcm-ECD in 37 (94.9%) patients. In control group, 38.3% ( 18/47) were induced epileptic seizures, including 77.8% (14/18) with subclinical seizures and 22.2% (4/18) with clinical seizures. The induction rate of epileptic seizures in clonidine plus sleep-deprivation group was significantly higher than that of control group (X2 = 13.614, P ＜ 0.01 ). However, there was no significant difference in clinical seizures between the two groups (X2 = 1.253, P ＞ 0.05 ). Conclusions The combination of oral intake of clonidine and sleep-deprivation could increase the induction rate of epileptic seizures and it is effective for epilepsy SPECT imaging.

The effects of Guizhi Fuling capsule and its active ingredient combination within different concentration on SPL proliferate were observed by MTT method. The ratio of CD80/86, CD3CD25 and CD3CD69 was used to evaluate cell activation effects of Guizhi Fuling capsule and its active ingredient combination by FCM. Guizhi Fuling capsule with concentration of 400 mg · L(-1)can promote spleen lymphocyte proliferation, as well as the active ingredient combination, which showed the obvious dose-effect relationship. Compared with control group, the difference has statistical significance (P≤0.01). The result of FCM showed that Guizhi Fuling capsule and its active ingredient combination can promote CD80 and CD86 expression on spleen lymphocyte, and also can increase CD25 and CD69 ratio between spleen CD3+ cells. Compared with control group, the difference has statistical significance (P≤0.01). Thus, Guizhi Fuling capsule and its active ingredient combination may have immune-modulate effects, and the mechanism may have a close relationship with the lymphocyte activation.
Animals ; Capsules ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Immunologic Factors ; pharmacology ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; drug effects ; immunology

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcriptional factors closely related to glucose and lipid metabolism, insulin sensitivity. Activation of PPARs targets treated type 2 diabetes, obesity, hypertension and other metabolic diseases by insulin resistance. Recently, a variety of active ingredients of traditional Chinese medicines (TCMs) have been proved to activate PPARs targets for improving insulin resistance, which has attracted widespread attention at home and abroad. In this paper, we reviewed the pathological mechanisms between insulin resistance and PPARs, and summarized the active ingredients of TCMs improved insulin resistance based on PPARs targets. This paper may provide some theoretical guidance for the development of new drugs and TCMs.
Animals ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Insulin Resistance ; Metabolic Diseases ; drug therapy ; genetics ; metabolism ; Peroxisome Proliferator-Activated Receptors ; antagonists & inhibitors ; genetics ; metabolism

Total RNA was extracted from leaf of Suaeda hetroptera kitag, then the CMO ( choline monooxygenase) cDNA was amplified using the reverse transcriptase polymerase chain reaction ( RT-PCR) method and cloned into pMD-T-simple vector. The positive clones from the Blue/White Screen were sequenced. After confirming its validity, the CMO gene fragment was cloned into pBI121 vector. Double enzyme restriction and PCR analysis indicated that the pBI121/CMO recombinant plasmid was successfully constructed.

The sfa1 gene encoded a bifunctional enzyme with the activities of both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase in Saccharomyces cerevisiae.The gene disruption cassette produced by PCR using the same long oligonucleotides which comprise 19 or 22 nucleotides complementary to sequences in the templates(pUG6 and pUG66 marker plasmid)at 3' end and 45 nucleotides at 5' end that annealed to sites upstream or downstream of the genomic target sequence to be deleted.After two linear disruption cassettes with a Cre/loxP mediated marker were transformed into the cells of Saccharomyces cerevisiae YS-1,the positive transformants were checked by PCR to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.Once correctly integrated into the genome,the select marker can be efficiently rescued by transformating the plasmid pSH47 into YS-1 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure.The expression of the Cre recombinase finally resulted in the removal of the marker gene,leaving behind a single loxP site at the chromosomal locus.The diploid mutant YS-1-sfa1 was generated,which could enhance the output of ethanol with 8.0% by shaking culture in flask compared with the original strain YS-1.

OBJECTIVE To investigate the risk factors,clinical characteristics and resistance of Staphylococcus aureus nosocomial infections so as to guide the treatment of S.aureus infection.METHODS To collect clinical materials of S.aureus nosocomial infection and analyze risk factors and clinical characteristics and detect sensitivity of isolated strains to antibacterial agents.RESULTS Severe underlying diseases existed among 73 cases of S.aureus nosocomial infections,82.19 percent of patients had received invasive interventions.Lower respiratory tract was the most common infective site.Seventy-nine strains of S.aureus were isolated,including 66 meticillin-resistant S.aureus(MRSA) and 13 meticillin-sensitive S.aureus(MSSA).S.aureus showed general resistance to many kinds of antibiotic drugs.The resistant rates of MRSA were much higher than those of MSSA(P