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Wei Liu

Publications and source records attributed to Wei Liu.

At least 325 records · Page 18Linked to original sources
PubMed2004

Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine: genetic approach to studying gene dose-dependent resistance to antifungals in A. fumigatus.

With the increasing use of antifungals such as amphotericin B, itraconazole, voriconazole, caspofungin, and terbinafine (TRB) in patients at high risk for invasive aspergillosis, resistance of Aspergillus fumigatus to these agents will ultimately emerge. Due to the limited availability of molecular genetics for A. fumigatus, few studies have addressed its mechanisms of resistance to antifungals. We transformed A. fumigatus protoplasts with a pyrG-based A. fumigatus genomic DNA library (constructed in the multicopy nonintegrating vector pRG3-AMA1-NotI, which also has the pyr-4 gene for selection). We obtained one pyrG(+) transformant that grew in medium containing a fungicidal concentration (0.625 microg/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid dependent, we evicted the plasmid and found concomitant loss of uracil prototrophy and TRB resistance. DNA sequence analysis identified the gene responsible for TRB resistance as the A. fumigatus squalene epoxidase gene (ERG1), which encodes the target enzyme of TRB. Authentic A. fumigatus ERG1, amplified from the genome and cloned into pRG3-AMA1-NotI, also conferred TRB-specific resistance. This molecular approach has the potential to enhance our knowledge of the mechanisms of A. fumigatus resistance to modern antifungals.

Amino Acid Sequence↗
PubMed2004

The infecting dose of Chlamydia muridarum modulates the innate immune response and ascending infection.

Murine vaginal infection with the obligate intracellular bacterium Chlamydia muridarum is commonly used as a model for ascending Chlamydia infections of the human female genital tract. Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity. However, many factors modify this response, including the bacterial load. To investigate the manner in which the inoculating dose of C. muridarum modulates a genital infection, we measured innate and adaptive cell numbers, CD4+ lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses of C. muridarum ranging from 10(4) to 10(7) inclusion-forming units. We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses. However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses. Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection. Taken together, these data suggest that innate immune cells contribute to control of ascending infection.

Animals↗
PubMed2004

Autoinduction of Bacillus subtilis phoPR operon transcription results from enhanced transcription from EsigmaA- and EsigmaE-responsive promoters by phosphorylated PhoP.

The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency. Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter. Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants. In vitro transcription studies using reconstituted EsigmaA, EsigmaB, and EsigmaE holoenzymes identified PA4 and PA3 as EsigmaA promoters and PE2 as an EsigmaE promoter. Phosphorylated PhoP (PhoP approximately P) enhanced transcription from each of these promoters. EsigmaB was sufficient for in vitro transcription of the PB1 promoter. P5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoP approximately P in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP approximately P at an EsigmaE promoter. Information concerning PB1 and P5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.

Bacillus subtilis↗
PubMed2004

Long-term SARS coronavirus excretion from patient cohort, China.

This study investigated the long-term excretion of severe acute respiratory syndrome-associated coronavirus in sputum and stool specimens from 56 infected patients. The median (range) duration of virus excretion in sputa and stools was 21 (14-52) and 27 (16-126) days, respectively. Coexisting illness or conditions were associated with longer viral excretion in stools.

Adult↗
PubMed2004

[Mutation and amplification of RIT1 gene in hepatocellular carcinoma].

OBJECTIVE: To explore the mutation and amplification of RIT1 gene and their correlation with carcinogenesis of hepatocellular carcinoma (HCC). METHODS: The polymerase chain reactioindirect sequencing method was used for detecting the mutations in the sequence of all 6 exons in the RIT1 gene of 50 HCC tissues and paratumor tissues. And the amplification of RIT1 gene was examined by fluorescence quantitative polymerase chain reaction method. RESULTS: A nucleotide 241 G --> C substitution in exon 5 of RIT1 gene was detected in one patient's HCC tissue, but not in paratumor tissue; this 241 G --> C substitution leads to Glu81Gln amino acid alteration in the conservative domain binding GTP. A nucleotide G --> C substitution in 5'-UTR (-21 bp from initial codon) was detected in all of the 50 HCC tissues and paratumor tissues, and 2- to 297-fold amplification of RIT1 gene was detected in 11 of 43 qualified cases, the amplification frequency being 25.6%. CONCLUSION: Gene amplification is one of the main activating ways of RIT1 gene in HCC, and its amplification might be correlated with HCC carcinogenesis, while point mutation might be not.

Adult↗
PubMed2004

[Experimental study on in vitro lamina propria engineering using oral fibroblast and polyglycolic acids].

PURPOSE: This study investigated the feasibility of lamina propria engineering in vitro using expanded oral fibroblast(OFC) and Polyglycolic Acids (PGA). METHODS: OFC were isolated by tissue explant method and expanded in vitro. OFC (20x10(6)) of 3rd passage were collected and then seeded onto PGA unwoven fibers to form a cell-scaffold. The constructs were cultured in DMEM +10% FBS. The cell-scaffold constructs were observed continuously by microscope. Small fragments were harvested at 1 week for electromicroscope, histological and RT-PCR analysis. RESULTS: At the sixth day, a neo-lamina propria was formed. HE and Masson stain revealed the formation of collagen fibers. RT-PCR revealed the new forming collagen was mainly type I collagen. CONCLUSION: lamina propria tissue is possible to engineer in vitro using oral fibroblast and polyglycolic acids. At this basis, we can construct bi-layer tissue engineering oral mucosa in the further research.

Fibroblasts↗
PubMed2004

[Study on the correlation between positive rates of SARS RNA in clinical confirmed SARS patients and the appearance of RNA in relation to the development of the disease].

OBJECTIVES: To study the correlation between positive rates of RNA in clinical confirmed severe acute respiratory syndrome (SARS) patients and its appearance in relation to the development of the disease in order to provide scientific basis for early diagnosis, effective prevention and treatment of the disease. METHODS: One-step reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the SARS RNA in the clinical specimens from different courses of the disease. The representative amplicons were then sequenced. Chi-square for trend test was performed to study the correlation between positive rates of RT-PCR and at different periods after the onset of the disease. RESULTS: The fragments amplified from the sputum specimens of SARS patients were shown to share 100% homology with the published SARS-associated coronavirus. Of the different clinical specimens, positive rate in the stools appeared to be the highest (21.55%). Chi-square for trend test revealed that the positive rates of stools and sputa of SARS patients decreased with the development of the disease (chi(2) for trend = 12.55 and 16.408, P = 0.0004 and P = 0.000 05 respectively). CONCLUSION: One-step RT-PCR proved to be an effective method for the detection of SARS-associated coronavirus from clinical specimens. Data as indicated that the positive rates of SARS coronavirus were decreasing in SARS patients along with the disease progression.

Adolescent↗
PubMed2004

[Development and evaluation of the kit for detection of SARS-associated Coronavirus RNA].

AIM: To develop a diagnostic kit for detection of SARS (severe acute respiratory syndrome)-related coronavirus RNA based on reverse transcription and polymerase chain reaction and to estimate its specificity and sensitivity. MATERIAL AND METHODS: 68 virus and bacterial cultures, 240 clinical samples from people without SARS symptoms and also 22 RNA samples from patients with SARS symptoms received during the epidemic in Beijing were used. RESULTS: The specificity of the kit was determined using animal coronaviruses and other bacterial and viral strains, causing acute respiratory and intestinal infections, and was shown to be 100%. The sensitivity of the kit in different clinical samples was 2.2 x 10(3) genome equivalents of recombinant SARS RNA in 1 ml of the specimen. The kit was evaluated in the Institute of Microbiology and Epidemiology of Beijing (China) using SARS-cov viral suspension and clinical samples from patients with suspected SARS. It was shown that kit was able to detect 10 TCID/50 ml of SARS-Cov virus. Testing of clinical samples from patients with suspected SARS showed that diagnostic sensitivity of the kit was 95%. Detection of the SARS-Cov RNA was more effective in feces compared to sputum 990 and 40%, respectively). CONCLUSION: The kit "AmpliSens SARS" for qualitative detection of SARS-related coronavirus RNA by reverse transcription and polymerase chain reaction (PCR) in nasopharyngeal wash/aspirates, naso/oropharyngeal swabs, plasma, and extract from feces has been developed in the Central Research Institute for Epidemiology of the RF Ministry of Health. The kit contains reagents for RNA isolation and purification, cDNA synthesis by reverse transcription of RNA, for PCR and for electrophoretic analysis of amplified products. The kit also contains recombinant positive and internal control samples allowing to control efficiency of analysis and showed good analytical and diagnostic characteristics.

Evaluation Studies as Topic↗
PubMed2004

[Trisomy 3 in intestinal B-cell lymphoma of mucosa-associated lymphoid tissue type].

OBJECTIVE: To determine the frequency of trisomy 3 in the intestinal B-cell lymphoma of mucosa associated lymphoid tissue (MALT) type and to explore the relationship between these two. METHODS: Paraffin embedded tissue sections of intestinal B-cell lymphomas of MALT type, according to WHO classification, were successfully studied. Seven cases were classical MALT lymphoma, being clinically low-grade, and one MALT lymphoma with large cell transform, clinically high-grade. By using biotin labelled DNA probes specific for chromosome 3 centromeric, chromosome in situ hybridization (CISH) were performed. Probe for C16 was used as the positive reference of the method, and chronic intestinal inflammation as the experiment control for detection of C3 copies in tumor cells. RESULTS: Of the 7 low-grade classical MALT lymphoma, 5 showed trisomy 3 and 2 normal C3. The only case of MALT transformed was trisomy 3. The frequency of trisomy 3 in the intestinal low-grade lymphoma was 71.4%. CONCLUSION: The frequency of trisomy 3 in the intestinal low-grade lymphoma is high, indicating that it may be involved in the pathogenesis of this disease and may be of value for the diagnosis.

Adult↗
PubMed2004

[In-vivo tracing of bone marrow stromal cell differentiating into chondrocytes by green fluorescent protein gene transfection].

AIM: To achieve highly efficient, stable and long-term expression of green fluorescent protein(GFP) reporter gene in bone marrow stromal cells(BMSCs) and to trace directly the distribution and differentiation of BMSCs in articular cartilage defects. METHODS: Recombinant RV-GFP expression vector was constructed and transfected into packaging cell PT67. After G418 screening and amplification, cell clones producing high level recombinant viruses were obtained and in-vitro expanded. The virus supernatant from infected PT67 cell culture was used to infect BMSCs directly. Autologous GFP-labeled BMSCs were seeded onto biodegradable polymer and implanted into porcine articular cartilage defects. 7 months later, the repaired tissue was evaluated by confocal microscope. RESULTS: The constructed recombinant expression vector RV-GFP was identified by restriction digestion. The transfection rates of PT67 cells reached 20%-50%, which reached 100% after G418 screening, bright green fluorescence could be seen under fluorescence microscope. BMSCs could be transfected successfully by the culture supernatant from infected PT67 cells and GFP was expressed efficiently in a long-term period. Confocal microscope revealed that GFP-labeled cells existed in many neocartilage lacunae after 7 months. CONCLUSIONS: The recombinant vector RV-GFP can provide a simple, sensitive and reliable tool to label BMSCs which can be used to study cytologic dynamic follow-up in-vivo. BMSCs can differentiate into chondrocytes and play an important role in repairing articular cartilage defects.

Animals↗
PubMed2004

[Relationship between expression of survivin and prognosis in human laryngeal squamous cell carcinoma].

BACKGROUND & OBJECTIVE: A novel inhibitor of apoptosis Survivin has recently been found in many common human cancers but not in normal tissues. Its potential distribution in human laryngeal squamous cell carcinoma (HLSCC) and its implication for inhibition of apoptosis were not yet very clear. This study was designed to investigate the expression of survivin and the probabilities of using it as a new indicator of the prognosis of HLSCC. METHODS: HLSCC samples from 71 patients treated in Department of Otorhinolaryngology, Second Affiliated Hospital of Sun Yat-sen University between 1995 and 1998 were analyzed for survivin expression by immunohistochemistry method. The patients were followed-up for long term and the relationship between survivin expression and clinical data including age, gender, classification, clinical type, pathological type, lymph node metastasis, distant metastasis, and prognosis were analyzed using software SPSS11.0. RESULTS: The expression rate was 50.7% (36/71) in all 71 cases. The expression rate was 81.8% (9/11) in the cases with lymph nodes metastasis, which was higher than that in the cases without lymph nodes metastasis (45%, 27/60), with significant difference (P=0.025). Univariate analysis revealed that classification,type and survivin expression were significantly related to the prognosis of HLSCC. P value were 0.0001, 0.009, and 0.0008, respectively. Multivariate analysis demonstrated that the patients with positive survivin had significant shorter survival (63.5 months) than those with negative survivin (84.0 months) (P=0.006) except the effect of classification and type on the prognosis of HLSCC. CONCLUSION: Survivin expressed in HLSCC especially in the cases with lymph nodes metastasis. Its expression may related to the prognosis of HLSCC.

Carcinoma, Squamous Cell↗
PubMed2004

[The detection of nanobacteria infection in serum of healthy Chinese people].

OBJECTIVE: Nanobacteria, a new kind of bacteria found by a Finnish scholar, is considered to relate to many human diseases like nephrolithiasis. However, there are no data available on nanobacteria infection in Chinese people. METHODS: Nanobacteria was detected in serum of 336 cases of healthy adults in Southern China by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry stain. The monoclonal antibody of nanobacterum was supplied by Kuipio University of Finland. RESULTS: Nanobacteria infection rates were 27 (8.0%), 19 (5.7%) in the healthy adults by ELISA and immunohistochemistry stain respectively. No difference was shown between the 2 methods and between male and female, statistically. Age and sex did not seem to be related to the infectious risk of nanobacteria. However, the infectious rate was lower in those below 30-year-old than that of people over 60-year-old (P < 0.05). CONCLUSION: Nanobacteria was existed in the serum of Chinese healthy people with an infectious rate of 8.0%.

Adult↗
PubMed2004

[Study on the molecular epidemiology of hantaviruse carried by hosts in northern suburb of Beijing].

OBJECTIVE: To investigate hantanvirus infection of captured rodents in Haidian district and Changping district of Beijing and to type hantavirus using molecular technique. METHODS: The captured mice were classified and the density of distribution was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify the partial M fragnments of hantaviruse. Several representative positive samples were sequenced and analysed by ClustalX (5.0) and DNAClub software. RESULTS: A total of 414 animals were captured, among which Battus norvegicus was the dominant group. In Haidian district, the median infection rates with hantavirus were 13.14% in Battus norvegicus and 0 in Mus musculus Linnaeus. In Changping district, the average infection rates were 17.46% in Battus norvegicus and 3.57% in Mus musculus Linnaeus. Nucleotide sequences analysis showed that the virus detected all belonged to SEO-type. They clustered with Z37 virus and could be branched into 2 different subclades. CONCLUSION: The major hosts of hantavirus in Haidian and Changping district were Battus norvegicus and the epidemic strains in the two districts of Beijing were genotyped as SEO-type. Nucleotide sequence and deduced amino acid sequence from different rodents were highly homologous, while nucleotide mutation had also been observed. Further studies are required to explore the possible virus sequence mutation.

Animals↗
PubMed2004

Activation of protein kinase A alters subnuclear distribution pattern of human steroidogenic factor 1 in living cells.

BACKGROUND: The aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation. METHODS: Full-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed. RESULTS: The transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA. CONCLUSIONS: Activation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor.

Cell Compartmentation↗
PubMed2004

[Regeneration of autologous tissue-engineered cartilage by using basic-fibroblast growth factor in vitro culture].

OBJECTIVE: To investigate the effect of the basic fibroblast growth factor (b-FGF) to regenerate an autologous tissue-engineered cartilage in vitro. METHODS: The Cells were harvested from the elastic auricular cartilage of swine,and were plated at the concentration of 1 x 10(4) cells/cm2 , studied in vitro at two different media enviroments: Group I contained Ham's F-12 with supplements and b-FGF, Group II contained Ham's F-12 only with supplements. The passage 2 cells (after 12.75 +/- 1.26 days) were harvested and mixed with 30% pluronic F-127/Ham's F-12 at the concentration of 50 x 10(6) cells/ml. It was injected subcutaneously at 0.5 ml per implant. The implants were harvested 8 weeks after the vivo culture and examined with the histological stains. RESULTS: The chondrocytes displayed morphologically similar to the fibroblasts in the media containing basic-FGF. The number of cell doublings (after 12.75 +/- 1.26 days) in vitro culture was as the following: Group I, 70; Group II, 5.4. Eight 8 weeks after the vivo autologous implantation, the average weight (g) and volume (cm3) in each group was as the following: Group I, 0.371 g/0.370 cm3 Group II, 0.179 g/0.173 cm3 (P < 0.01). With the b-FGF in vitro culture, the cells were expanded by 70 times after 2 weeks. Histologically, all of the engineered cartilage in the two groups were similar to the native elastic cartilage. CONCLUSION: These results indicate that the basic-FGF could be used positively to enhance the quality and quantity of the seeding cells for the generation of the well-engineered cartilage.

Animals↗
PubMed2004

[Reconstruction of rabbit corneal stroma using tissue engineering technique].

OBJECTIVE: Reconstruct corneal stroma by tissue engineering. METHODS: Primary corneal stromal cells were isolated from newborn rabbit cornea. When the cultured cells reaching confluence, the stromal cells were mixed with polyglycolic acid (PGA) to form a cell-scaffold construct. After 1 week in vitro culture, the constructs were implanted into mother rabbit corneal stroma. Parts of corneal stromal cells were transfected with GFP gene as mark of transplanted cells. Tissues were harvested at 8 weeks for transmission electron microscopy (TEM), histology and Western blot evaluation. In control, PGA alone was implanted into the other cornea. RESULTS: The engineered corneal stroma became transparent gradually over a period of 8 weeks. The histology of engineered stromal lamellar was relatively similar to that of natural one, no significant differences were found in the diameter of cornea collagen fiber [(29.4 +/- 4.7) nm] in experimental rabbits compared with control [(28.5 +/- 3.5) nm], Student's t-test: P = 0.1316 > 0.05. TEM demonstrated that collagen fibrils deposited in engineered stroma had a similar diameter compared to that of normal counterpart. In addition, Western blot showed the positive expression of type I collagen in the collagen fibrils. In contrast, no new stroma tissue was formed when PGA alone implanted. A green colored stroma was observed when engineered with GFP-labeled cells under fluorescence light microscope. CONCLUSION: The results demonstrate that nearly transparent corneal stroma can be obtained by the technique of cornea engineering.

Animals↗