Objective To analyze the sensitivity of clinical isolates of Candida albicans to flucona- zole,to detect mutations in their ERG11 genes,and to investigate the correlation between ERG11 gene mutation and resistance to fluconazole.Methods Candida albicans was identified from clinical isolates of Candida spp..The sensitivity to fluconazole was detected in vitro by microdilution-basesd method and Rosco tablets method.Three pairs of primers were designed to amplify three fragments of ERG11 gene(483 bps, from 295 bp to777 bp;482bps,from 723 bp to 1204 bp;489 bps,from 1179 bp to 1667 bp)after the extracting of genomic DNA.PCR products were sequenced.Results Eighty clinical isolates of Candida spp.were collected,which included 52 isolates of Candida albicans,all of which were sensitive to flucona- zole.Nineteen mutations were detected in ERG11 gene of 5 fluconazole-sensitive clinical isolates.Of the 19 mutations,14 were samesense mutations,and the remaing 5 missense mutations(T495A,A530C, G640A,A945C and G1609A),resulting in amino acid substitution D116E,K128T,E165K,E266D and V488I,respectively in lanosterol 14 alpha-demethylase.E165K was a novel mutation.Conclusions The clinical isolates of Candida albicans were highly sensitive to fluconazole;E165K and V488I might not lead to the resistance of Candida albicans to fluconazole.

OBJECTIVETo study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).

METHODS(1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.

RESULTS(1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).

CONCLUSIONSTIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.

Calcium ; metabolism ; Cell Adhesion Molecules ; physiology ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; Nitric Oxide ; metabolism ; Stromal Interaction Molecule 2 ; TRPC Cation Channels ; physiology

OBJECTIVETo investigate the toxicological mechanism of microcystin-LR (MCLR) on L-02 cells.

METHODSL-02 cells was treated with MCLR at different concentrations and the subsequent changes such as cell proliferation (MTT assay), morphology, lactate dehydrogenase (LDH) leakage, apoptosis rate and apoptosis-related gene expression were examined.

RESULTSMTT assay showed that MCLR mildly inhibited the cell growth within the initial 24 h of treatment but enhanced the cell viability after that till 60 h in a time- and dose-dependent manner. LDH leakage underwent no marked changes in response to 48-hour MCLR treatment but increased upon prolonged treatment for 60 h, indicating the presence of oxidative damage. After a 48-h treatment with MCLR at 50 microg/ml, obvious apoptosis of L-02 cells occurred as manifested by cell rounding, detachment from the substrate, cell shrinkage and membrane blebbing. The apoptosis rates were rather low (between 22% and 29%) after treatment with MCLR at different concentrations for 36 h, and increased to as much as 80% after a 60-h treatment with 50 microg/ml MCLR. The expressions of p53 and bcl-2 increased in the cells after treatment with high-concentration MCLR, suggesting that MCLR up-regulated the expression levels of the two proteins.

CONCLUSIONMCLR can induce apoptosis and up-regulate p53 and bcl-2 expressions in human normal liver cell line L-02.

Apoptosis ; drug effects ; Cell Line ; Hepatocytes ; cytology ; Humans ; Microcystins ; toxicity ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

AIM:To clarify whether sulforaphane (SF) has protective effects on retina neuronal cells in dia-betic rats and to identify the related mechanisms involved in this process. METHODS:The diabetic rat model was induced by single intraperitoneal injection of streptozotocin (STZ). The protective effects of SF were evaluated by measuring the generation of reactive oxygen species(ROS),detecting apoptosis of retina neuronal cells with TUNEL staining and counting the survival retinal ganglion cells (RGCs). The nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) and the protein expression of heme oxygenase-1 (HO-1) were examined by immunofluorescence analysis and Western blot. RE-SULTS:SF treatment significantly attenuated ROS generation, decreased the apoptosis of retina neuronal cells and in-creased the numbers of survival RGCs in the diabetic rats. Meanwhile,SF significantly increased the nuclear accumulation of Nrf2 and the protein level of HO-1 in the retinas of diabetic rats. However,HO-1 inhibitor,protoporphyrin Ⅸ zinc(Ⅱ) diminished the inhibitory effects of SF on RGCs apoptosis. CONCLUSION:SF partially exerts the beneficial neuroprotec-tive effects via the activation of the Nrf2/HO-1 antioxidant pathway,therefore alleviating retinal oxidative stress and decrea-sing the apoptosis of retina neuronal cells.

OBJECTIVETo verify the clinical efficacy of heat-sensitive moxibustion in treatment of knee osteoarthritis (KOA).

METHODSSixty cases of KOA were randomly divided into a heat-sensitive moxibustion group and a conventional moxibustion group, 30 cases in each one. Dubi (ST 35), Yanglingquan (GB 34), Zusanli (ST 36) and Heding (EX-LE 2) on the affected side were selected in two groups. In heat-sensitive moxibustion group, the techniques of circling moxibustion, sparrow-pecking moxibustion, moving moxibustion and mild moxibustion were applied. In conventional moxibustion group, the mild moxibustion was used, 2 to 3 cm far from the skin of the acupoints selected. Lysholm scale for the assessment of knee joint function was adopted to evaluate the efficacy. The scores of joint pain, morning stiffness, joint swelling and walking ability were compared before and after treatment in two groups.

RESULTSThe scores of joint pain, morning stiffness, joint swelling and walking ability after treatment were all apparently improved as compared with those before treatment in either group (all P < 0.05). The improvement in the above-mentioned indices in heat-sensitive moxibustion group was much more apparent as compared with that in conventional moxibustion group (all P < 0.01). The effective rate was 90.0% (27/30) in heat-sensitive moxibustion group and was 73.3% (22/30) in conventional moxibustion group. The effective rate in heat-sensitive moxibustion group was obviously superior to that in conventional moxibustion group (P < 0.01).

CONCLUSIONThe efficacy of heat-sensitive moxibustion is superior to that of conventional moxibustion in the treatment of KOA. This therapy can more significantly improve the symptoms and physical signs of the patients with KOA.

Acupuncture Points ; Aged ; Female ; Humans ; Locomotion ; Male ; Middle Aged ; Moxibustion ; Osteoarthritis, Knee ; physiopathology ; therapy ; Treatment Outcome

OBJECTIVETo investigate the genetic association between schizophrenia and polymorphism of D-amino acid-oxidase (DAAO) gene.

METHODSA total of 112 parent/offspring trios in which the proband met the Amerecan Classification and Diagnostic Criteria for Mental Disorders (Fourth Revised Edition) were included in this study. Correlation analysis between schizophrenia and DAAO gene polymorphism and haplotype relative risk analysis were conducetd by using PCR and SNP typing in all the nuclear families.

RESULTSThe rs3918347 allele was correlated to schizophrenia (P = 0.014). Allele A was a protective factor (Z = -2.37) and allele G the hazard factor (Z = 2.37). The frequency of rs3918347 allele A was 0.41 and that of the allele G was 0.59. The rs3741775, rs3825251 and rs4964770 alleles were not associated with schizophrenia. Three haplotypes of C/G in the rs3825251-rs3918347, G/T in the rs3918347-rs4964770, C/G/T in the rs3825251-rs3918347-rs4964770 were associated with schizophrenia (P = 0.021, 0.036, and 0.028, with genotype frequencies of 0.33, 0.28, and 0.15, respectively).

CONCLUSIONThe nucleotide polymorphism of DAAO gene is associated with schizophrenia in Chinese population.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Carrier Proteins ; genetics ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Schizophrenia ; genetics ; Young Adult

BACKGROUNDMacrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study, we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM).

METHODSHuman bone marrow CD34(+) cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively.

RESULTSMSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys + EC-CM, Cys + MSP or Cys compared with 0 hour control in liquid culture system after 6 days.

CONCLUSIONMSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

Antigens, CD34 ; analysis ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chemokine CCL3 ; Chemokines, CC ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Proto-Oncogene Proteins ; pharmacology

OBJECTIVE: To observe the effects of endothelial cells from umbilical cord blood (UCB) on the amplification of human early hematopoietic cells from UCB in vitro. METHODS: Endothelial cells from UCB were cultured by the optimized medium of endothelial cells. There were 2 experiment groups: cytokines group (SCF+IL-3+IL-6+GM-CSF, CKs group) and noncontact group (endothelial cell layer with CKs without contacting the CD34+ cells group). CD34+ cells from UCB were isolated by MiniMACS. After the cells in the CKs group and the noncontact group were cultured for 7 days, the amplifying folds of early hematopoietic cells were assayed. RESULTS: Early hematopoietic cells from UCB were expanded in the CKs group or the noncontact group. The amplifying folds of the noncontact group on early hematopoietic cells were significantly more than those of the CKs group. CONCLUSION The amplification effect of the noncontact group on early hematopoietic cells is superior to that of the CKs group.
Antigens, CD34 ; analysis ; Cell Proliferation ; drug effects ; Coculture Techniques ; Culture Media ; pharmacology ; Cytokines ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Fetal Blood ; cytology ; metabolism ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Immunohistochemistry

OBJECTIVE: To explore the characteristic changes of the peripheral chorda tympanic nerve (CT) electrophysiological responses to salty stimulus and other taste stimuli in rats with the conditioned taste aversion to saltiness. METHODS: Fourteen adult SD male rats were divided into a conditioned taste aversion to salty group (CTA) and a control group (Ctrl) (n=7/group). On the first day of the experiment, rats were given a 0.1 mol/L NaCl intake for 30 min, then, the rats in CTA and Ctrl groups were injected intraperitoneally with 2 ml of 0.15 mol/L LiCl and the same amount of saline respectively. On day 2, 3 and 4, the 30 min consumption of NaCl and distilled water was measured for both groups of rats. On the 4th day after the behavioral test of that day, CT electrophysiological recording experiments were performed on CTA rats and control rats. RESULTS: Compared with the rats in Ctrl group, the electrophysiological characteristics of CT in CTA group rats did not change significantly the responses to the series of NaCl and other four basic taste stimuli (P＞0.05). The amiloride, the epithelial sodium channel blocker, strongly inhibited the response of CT to NaCl in CTA and Ctrl group rats (P＜0.01). CONCLUSION The electrophysiological responses of CT to various gustatory stimuli do not significantly change in rats after the establishment of conditional taste aversion to the saltiness.
Amiloride ; pharmacology ; Animals ; Chorda Tympani Nerve ; physiology ; Conditioning, Classical ; Electrophysiological Phenomena ; Male ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride ; Taste ; physiology

Objective To study the biological macrofeatures and criterion thereof for fracture immobilization in Chinese Mongolian traditional osteopathy. Method The principles and methods of modern physiological psychology and biomechanics were used in this study to explore the biological macrofeatures and criterion thereof for fracture immobilization, based on the view of the harmony of human and nature (including a unity of body and function) in Chinese Mongolian traditional osteopathy. Results Chinese Mongolian traditional osteopathy implies the biological macrofeatures and criterion thereof for “dynamic immobilization” in fracture treatment, including the stability of structure and force catched, state of static and dynamic, forming and destroying of bone, physical and psychological stability. Therefore, it is a kind of non invasive and non shelter fixing method. Conclusions The biological macrofeature and criterion thereof for the fracture dynamic immobilization in fracture treatment, in Chinese Mongolian traditional osteopathy is not only the fundamental support for its inheritance up to now, but also could be a new attempt in modern fracture immobilization.