Activated protein C(APC)is generated through hydrolysis of protein C by thrombin-thrombomodulin complex.APC has been proved to play different roles in many human diseases,such as anticoagulant,antiinflammatory,profibr-inolysis,antiapoptotic and endothelial cytoprotective activities.In this review,the research progress of the functions of APC and related diseases,especially the neural protective roles of APC in central nervous system were summarized.

OBJECTIVETo study the molecular types of Staphylococcus aureus isolated from a severe food-poisoning and to trace the possible strains.

METHODSReal-time PCR was applied to detect nuc gene as a specific marker for S. aureus, mecA gene encoding methicillin resistance and 5 other genes encoding staphylococcal enterotoxins (sea, seb, see, sed, see). Isolates were also performed with 16S rRNA oligonucleotide sequence analyzing by DNAStar MegAlign 5.0 software and pulse-field gel electrophoresis (PFGE) by BioNumerics Version 4.0 software.

RESULTSThe nuc gene was detected from the 10 isolated strains, sea and seb genes were detected from 7 strains. There were 4 16 S rRNA types and 5 PFGE types found from all the strains.

CONCLUSIONSThree relative S. aureus strains were involved in the severe food-poisoning at least. Molecular subtyping might give a molecular epidemiological evidence and support the source tracing of an outbreak.

Bacterial Typing Techniques ; China ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxins ; Humans ; Staphylococcal Food Poisoning ; epidemiology ; microbiology ; Staphylococcus aureus ; classification ; genetics ; isolation & purification

OBJECTIVETo construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction.

METHODSThe fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting.

RESULTSEnzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent.

CONCLUSIONThe cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.

Animals ; Blotting, Western ; COS Cells ; Cell Line ; Cell Membrane ; metabolism ; Cell Nucleus ; metabolism ; Cercopithecus aethiops ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Mice ; Microscopy, Fluorescence ; NIH 3T3 Cells ; Nuclear Localization Signals ; genetics ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo apply pulse-field gel electrophoresis analysis(PFGE) in analysing a case of food poisoning caused by Vibrio parahaemolyticus.

METHODSPFGE using restriction enzyme Not I was employed in molecular subtyping of thirty strains of V. parahaemolyticus isolated from a case of food poisoning in Guangzhou city and PFGE patterns were analyzed by using BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by using the Dice coefficient and unweighted pair group method with arithmetic averages (UPGMA).

RESULTSThirty strains were of the same type of pulsotype.

CONCLUSIONSMolecular subtyping by PFGE might disclose the epidemiological relationships of the strains from humans, food and the environment, giving a strong molecular epidemiological evidence and a support for the source-tracking of outbreak events.

Bacterial Typing Techniques ; methods ; China ; Electrophoresis, Gel, Pulsed-Field ; methods ; Foodborne Diseases ; microbiology ; Humans ; Vibrio parahaemolyticus ; classification ; genetics ; isolation & purification

OBJECTIVETo observe the therapeutic effects between abdominal acupuncture and Deanxit for treatment of menopause depressive disorder, and to explore the efficacy and safety of abdominal acupuncture.

METHODSSixty cases were randomly divided into an observation group and a control group, 30 cases in each. The observation group was treated with abdominal acupuncture at Zhongwan (CV 12), Xiawan (CV 10), Qihai (CV 6) and Guanyuan (CV 4), etc. The control group was treated with oral administration of Deanxit. The patients in both groups were treated for 4 weeks and followed up for another 4 weeks, and they were evaluated by Hamilton Depression Scale (HAMD) every couple weeks.

RESULTSThe total HAMD scores of 2 and 4 weeks treatments and 2 and 4 weeks follow-up were all reduced in both groups (all P < 0.01). The total scores of 2 and 4 weeks follow-up in observation group were lower than those in control group, with significant differences in statistical analysis (both P < 0.05). Compared with the clinical therapeutic effect of both groups after 4 weeks treatment, there was no significant difference (P > 0.05), however, after 4 weeks follow-up, the therapeutic effect in observation group was superior to that in control group, with significant difference in statistical analysis (P < 0.05). The safety indexes before and after treatment of both groups were normal, and the adverse reaction rate in observation group was much lower than that in control group (P < 0.05).

CONCLUSIONAbdominal acupuncture is an effective and safe method for menopause depressive disorder, it improves the menopause depressive symptoms with persistent action, less symptoms relapse and adverse reactions.

Abdomen ; Acupuncture Therapy ; Adult ; Anthracenes ; therapeutic use ; Depressive Disorder ; drug therapy ; therapy ; Drug Combinations ; Female ; Flupenthixol ; therapeutic use ; Humans ; Menopause ; drug effects ; psychology ; Middle Aged

OBJECTIVETo study dynamics of T lymphocyte subsets in severe acute respiratory syndrome (SARS).

METHODSSequential anti-coagulated blood samples were collected from 46 cases of SARS patients during the 1st week, the 2nd week, the 3rd-5th week and the 8th-12th week after the infection. T lymphocyte subsets including CD3+CD4+ cells, CD3+CD8+ cells, naive CD4+ cells (CD4+CD45RA+CD62L+) and activated CD8+ cells (CD8+CD38+) were detected by 3-color flow cytometry. Fifty-six normal healthy blood donors were also detected as normal controls.

RESULTSCompared with the results of normal controls, both of the percentages of CD4+ cells and CD8+ cells of SARS patients were in normal levels during the 1st week, but the cell counts decreased significantly to (306 +/- 140)/mm3 and (270 +/- 143)/mm3, respectively. The cell count of naive CD4+ subset also remarkably decreased to (96 +/- 49)/mm3, and the percentage of CD8+CD38+ subset was higher than that of normal controls [(59.3 +/- 12.6)% vs (44.9 +/- 12.5)%]. During the 3rd-5th week, the CD8+ cell count and the percentage of CD8+CD38+ subset reached normal values, which were (581 +/- 356)/mm3 and (40.1 +/- 17.6)%, respectively. During the 8th-12th week, the cell counts of CD4+ cell [(578 +/- 193)/mm3] and naive CD4+ subset [(176 +/- 64)/mm3] were still less than those of normal controls, while compared with those of the 1st week, the increments were remarkable.

CONCLUSIONST lymphocytes of SARS patients decreased dramatically but could be obviously resumed in a short time. It will take more than 8-12 weeks for CD4+ cell and naive CD4+ subset to reach to normal levels.

Adult ; CD4 Lymphocyte Count ; CD4-CD8 Ratio ; CD4-Positive T-Lymphocytes ; immunology ; Female ; Humans ; Male ; Middle Aged ; Severe Acute Respiratory Syndrome ; immunology ; T-Lymphocyte Subsets ; immunology

OBJECTIVETo explore the clinicopathological correlation between CD4(+) T lymphocyte count and superficial lymphadenopathy HIV/AIDS patients.

METHODSA total of 1066 HIV/AIDS patients were included in this study. The incidence of superficial lymphadenopathy, peripheral blood CD4(+) T lymphocyte counts and histological features of superficial lymphadenopathy were analyzed.

RESULTSAmong 1066 patients, 126 cases (11.8%) presented with superficial lymphadenopathy. Of the 126 cases, there were 69 cases with CD4(+) T lymphocyte counts < 100/µl and clinical diagnoses including tuberculosis (37 cases), reactive hyperplasia (8 cases), AIDS-related lymphadenopathy (18 cases), penicillium diseases (12 cases), fungal infection (5 cases) and non-tuberculous mycobacterial infection (1 case). Twenty-six cases had CD4(+) T lymphocyte counts between 100/µl to 200/µl and clinical diagnosis including tuberculosis (12 cases), reactive hyperplasia (8 cases), AIDS-related lymphadenopathy(6 cases), penicillium disease (2 cases) and non-Hodgkin lymphoma (1 case). Twenty-nine cases had CD4(+) T lymphocyte counts > 200/µl and clinical diagnoses including tuberculosis (11 cases), reactive hyperplasia (12 cases), AIDS-related lymphadenopathy (3 cases), Penicillium diseases (1 case) and non-Hodgkin lymphoma (4 cases). The CD4(+) T lymphocyte counts among patients with tuberculosis, AIDS-related lymphadenopathy and Penicillium diseases were significantly different (χ(2) = 8.861, P = 0.012). A significant correlation between the incidence of superficial lymphadenopathy and CD4(+) T lymphocyte counts was found (χ(2) = 375.41, P = 0.000).

CONCLUSIONSThe most common cause of superficial lymphadenopathy in HIV/AIDS patients is tuberculosis, followed by lymph node reactive hyperplasia, AIDS-related lymphadenopathy and Penicillium disease. Low CD4(+) T lymphocyte count correlates with an increased incidence of superficial lymphadenopathy and the risk of opportunity infection. Therefore, determination of peripheral blood CD4(+) T lymphocyte count should become an integral marker for the early diagnosis and treatment of superficial lymphadenopathy in HIV/AIDS patients.

AIDS-Related Complex ; blood ; complications ; pathology ; AIDS-Related Opportunistic Infections ; blood ; complications ; pathology ; Acquired Immunodeficiency Syndrome ; blood ; complications ; pathology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; CD4 Lymphocyte Count ; Child ; Female ; HIV Infections ; blood ; complications ; pathology ; Humans ; Lymph Nodes ; pathology ; Male ; Middle Aged ; Tuberculosis ; blood ; complications ; pathology ; Young Adult

OBJECTIVETo explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.

METHODSThe Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.

RESULTSpET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.

CONCLUSIONThe prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.

Amino Acid Sequence ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; HIV-1 ; genetics ; Humans ; Microscopy, Fluorescence ; Molecular Sequence Data ; Protein Transport ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism ; Transfection ; methods ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

OBJECTIVETo apply pulse-field gel electrophoresis analysis(PFGE) in the analysis of cholera outbreak events and to determine the molecular epidemiological characteristics of Vibrio cholerae ( V. cholerae) isolates.

METHODSPFGE using restriction enzyme Not I was employed in the molecular subtyping of forty-one strains of V. cholerae isolated in cholera outbreak events from 2003 to 2005 in Guangzhou area and PFGE patterns were analyzed by BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by utilizing of Dice coefficient and UPGMA(unweighted pair group method with arithmetic averages). Comparison of PFGE typing results was performed with phage-biological typing and pathogenicity-associated genes typing.

RESULTSIn cholera outbreak events, PFGE could discriminate epidemiologically related and unrelated strains, having more discriminatory power than phage-biological typing and pathogenicity-associated genes-typing.

CONCLUSIONSMolecular sub-typing by PFGE could disclose the epidemiological relationships of strains from humans and the environment, providing molecular epidemiological evidence and support for the source-tracking of cholera outbreak events.

Bacterial Typing Techniques ; methods ; Cholera ; epidemiology ; microbiology ; Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Molecular Epidemiology ; Vibrio cholerae ; classification ; genetics ; isolation & purification

OBJECTIVETo apply multiplex polymerase chain reaction (MPCR) assay and sequencing in study of the carrying status of four pathogenicity-related genes of Vibrio cholerae (V.cholerae) and the variation of ctxA.

METHODSPrimers targeting cholera toxin sub-unit A gene (ctxA), toxin-coregulated pilus gene (tcpA), accessory cholera enterotoxin gene (ace), zonula occludens toxin gene (zot) were designed and the MPCR method was applied to detect the pathogenicity-related genes of 276 strains of V.cholerae isolates. The amplified fragments of ctxA gene were sequenced and the genetic homology of the amplified fragments of ctxA was analyzed.

RESULTSOf the 276 strains of V.cholerae, 93.9% strains from human sources belong to the pathogenicity-related genes type A (ctxA(+)tcpA(+)ace(+)zot(+) type) and 6.1% belong to pathogenicity-related genes type C (ctxA(-)tcpA(-)ace(-)zot(-) type). Type A strains from clinical sources were isolated from patients with mild to severe symptom and carriers, among which 68.5% were isolated from patients with mild symptom and 21.9% from carriers. All 63.6% of type C strains from clinical sources were isolated from patients with mild symptom and 36.4% from carriers. The proportion of type C strains that caused mild symptom was higher than that of type A strains. Of the 78 strains isolated from the environment, 9.0% strains belong to pathogenicity-related type A and 35.9% belong to the pathogenicity-related genes type B (ctxA(-)tcpA(-)ace(+)zot(+) type), while 55.1% belong to pathogenicity-related genes type C. The sequencing results showed little genetic variation among the amplified fragments for ctxA.

CONCLUSIONMPCR disclosed the polymorphic status of pathogenicity-related gene patterns in V.cholerae isolates of Guangzhou, providing effective means for further study on evolution of pathogenicity-related genes among V.cholerae isolates from human and environmental sources. This study also offers significant guidance for effective prevention, control and warning against cholera epidemic in local area.

China ; Cholera Toxin ; genetics ; DNA, Bacterial ; Genes, Bacterial ; genetics ; Genotype ; Humans ; Polymerase Chain Reaction ; Sequence Analysis ; Vibrio cholerae ; classification ; genetics ; isolation & purification