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Biomedical subjects

B Feng

Publications and source records attributed to B Feng.

At least 37 records · Page 2Linked to original sources
PubMed2000

ASD4, a new GATA factor of Neurospora crassa, displays sequence-specific DNA binding and functions in ascus and ascospore development.

A new gene encoding a novel GATA factor, ASD4, of Neurospora crassa was isolated and demonstrated to possess one intron and to specify an open reading frame encoding a protein with 427 amino acid residues. The ASD4 protein contains a single GATA-type zinc finger and a putative coiled-coil domain. Unlike related proteins, DAL80 in yeast and NREB in Penicillium, ASD4 does not appear to be involved in regulation of nitrogen metabolism. An Asd-4 null mutant obtained by the rip procedure did not show any effect upon nitrogen control, but instead resulted in severe defects in ascus and ascospore genesis. The Asd-4 rip mutant is dominant to Asd-4+. A cross of the Asd-4 mutant with wild-type resulted in fruiting bodies that appeared to be normal macroscopically but which were complete devoid of asci and ascospores. Introduction of the Asd-4+ gene into the Asd-4 rip mutant corrected the defect in ascus and ascospore development in crosses with wild-type. Mobility shift assays demonstrated that ASD4 acts as a sequence-specific DNA binding protein and recognizes DNA fragments that contain GATA core elements. Gel filtration and cross-linking experiments revealed that the ASD4 protein exists as a tetramer in solution. These results suggest that the ASD4 protein functions positively as a transcriptional regulator of sexual development in Neurospora.

Blotting, Northern↗
PubMed2000

[Effects of cadmium chloride on the malignant transformation of human embryo lung fibroblasts].

The malignant transformation of human embryo lung fibroblast (HELF) induced by CdCl2 was tested by 3H-TdR incorporation, chromosome analysis and flow cytometry. Chromosome aberration, the ratio of cells in different cell cycle phases and the quantities of DNA synthesis of different groups were determined. The results showed that the morphology of HELF changed in three levels of cadmium chloride (I 0.004 mumol/L, II 0.02 mumol/L and III 0.04 mumol/L) treated for three times. The cell growth was disordered and overlapping without density regulating effect and the transforming foci were formed. The quantities of chromosome were decreased or increased and the dicentric and acentric fragments were found in experimental groups. The ratio of cells in S phase increased and the quantities of DNA synthesis in experimental groups were 2.4-3.8 times higher than that of negative control group. The chromosome aberration of cells, the increase of cells in S phase and the increase of DNA synthesis may play some roles in the malignant transformation of HELF induced by CdCl2.

Cadmium Chloride↗
PubMed2000

A simple nanoelectrospray arrangement with controllable flowrate for mass analysis of submicroliter protein samples.

A simple arrangement for nanoelectrospray ionization using a conventional syringe pump connected to a pulled unmodified capillary has been evaluated. This arrangement avoids several disadvantages associated with metal-coated nanoelectrospray emitters. The relatively large orifice (approximately 9 microns) at the pulled capillary tip reduces sample clogging and the use of the pump minimizes spray disruption due to gas bubbles. Subattomole detection limit was achieved with nanomolar protein sample solutions at 5-10 nL/min flowrates using an LCQ mass spectrometer. Submicroliter samples can be loaded from the tip orifice and stored inside the capillary to virtually eliminate any dead volume, and then be electrosprayed for extended periods at well-controlled flowrates.

Mass Spectrometry↗
PubMed2000

Electrospray ionization Fourier transform ion cyclotron resonance analysis of large polymerase chain reaction products.

In previous work, we demonstrated the use of electrospray ionization to analyze small differences in size or sequence of relatively small polymerase chain reaction (PCR) products of 114 base pairs or less. The sequence information required to answer a biological question may be only a single nucleotide substitution or deletion. In many cases, the regions where these sequence variations can occur are several hundred base pairs in length, and the analysis of large PCR products is therefore desirable. Therefore, we have attempted to expand the size range of PCR products that can be analyzed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Previous work has shown that the difficulties associated with PCR product analysis increase with product size. A revised cleanup scheme was employed to target the removal of detergents with ethanol wash or precipitation steps, followed by additional desalting. Additionally, an in-trap cleanup to collisionally induce dissociation of noncovalent salt adducts was employed. This approach was extended to a 223 base pair PCR product yielding mass measurement accuracy within 26 ppM. The mass measurement accuracy obtained illustrates that a single base substitution could be identified at this size of PCR product with a 7 tesla ESI-FTICR.

Bacteriophage lambda↗
PubMed2000

Intracellular processing and toxicity of the truncated androgen receptor: nuclear congophilia-associated cell death.

The pathogenesis of the selective motor neuron death in spinal bulbar muscular atrophy (SBMA) is not fully understood. Similar to observations with other mutant polyglutamine (poly Q) expanded proteins, truncated androgen receptor (AR) with expanded poly Q tract cause intracellular aggregates; however, the precise relationship between aggregates and disease pathogenesis is unresolved. In order to have a better understanding of the cellular processing and toxicity of the mutant AR, we focused on a short N-terminal portion of AR containing normal or expanded poly Q repeats, and have carried out biochemical, immunocytochemical, cytochemical and ultrastructural studies of BHK cells at different intervals after transfection. In cells expressing mutant truncated AR, using an anti-AR N-terminal antibody, we observed no immune staining in the nucleus and identified immune negative aggregates surrounded by immunopositive material in the cytoplasm. Congo red staining identified a component of aggregates with a beta-pleated secondary structure in both cytosol and nucleus, while electron microscopy revealed a fibrillary-granular material as the ultrastructural correlate. In addition, acid phosphatase staining and ubiquitin immunocytochemistry demonstrated that in transfected cells, both lysosomal and nonlysosomal degradation systems are actively involved in handling the mutant truncated AR. The temporal relationship of nuclear congophilia to a subsequent massive cell death suggests that entry of proteolytic cleavage products into the nucleus, perhaps the expanded poly Q stretch itself, may play an important role in cell toxicity.

Animals↗
PubMed2000

Development of mouse dendritic cells from lineage-negative c-kit(low) pluripotent hemopoietic stem cells in vitro.

Dendritic cells (DCs) are essential for the presentation of antigens in the primary immune response. To examine the generation of DCs from hemopoietic stem cells in the bone marrow (BM), lineage-negative (Lin-)/CD71- bone marrow cells (BMCs) from C57BL/6 mice were separated into major histocompatibility complex (MHC) class Ihigh/ c-kit(low) and MHC class Ihigh/c-kit(low)(phenotypically c-kit-negative, but c-kit message only detected by reverse transcriptase-polymerase chain reaction) populations. A large number of cells with the morphological, phenotypical, and functional characteristics of DCs was generated from both c-kit(low) and c-kit(low) populations when cultured with a combination of cytokines (GM-CSF, tumor necrosis factor-a [TNF-a], interleukin 7 [IL-7], IL-3, stem cell factor [SCF], and flt3 ligand); the cytokine combination studies revealed that SCF and IL-3 in addition to GM-CSF and TNF-a are essential for DCs to be generated from these primitive populations. To our surprise most (>80%) generated cells expressed high levels of DC surface markers such as DEC205 and MHC class II, and they were potent stimulators in the primary allogeneic T cell activation. The development of DCs from c-kit(<low) cells was slower than that from c-kit(low) cells. These results indicate that c-kit(<low) cells are more primitive than c-kit(low) cells, although both c-kit*(low) cells and c-kit(<low) cells can differentiate into DCs. It should be noted that the combination of these cytokines selectively induces DCs from both c-kit(<low) and c-kit(low) cells in vitro, suggesting that the ex vivo expansion of DCs using these primitive cells would be applicable to immunotherapy.

Animals↗
PubMed2000

[Relationship between factor v Leiden mutation and Chinese Budd-Chiari syndrome and its clinical significance].

OBJECTIVE: To investigate the relationship between factor v Leiden (FvL) mutation and Chinese sporadic Budd-Chiari syndrome (BCS), familial BCS, and to explore the significance of FvL mutation in the etiology of BCS. METHODS: Twenty-five patients with sporadic BCS, 6 patients with familial BCS (from A and B families), 39 both A and B family members, and 31 healthy persons were detected for FvL mutation with PCR. Meantime, two family members were explored for the related etiology of BCS. RESULTS: Factor V Leiden mutation was detected in 4 of 6 patients with familial BCS and in 2 family members. AIII(7,11,15) and BII(10), AII(2) and BIII(5) were found FvL mutation, and mutation was heterzygous. FvL mutation in the two degrees was compatible with Mendel hereditery law. The frequency of FvL mutation in 31 BCS and 31 healthy persons showed no statistical significance: but the frequency of FvL mutation between the familial BCS and healthy persons showed statistical significance. Ten persons in A family had varicose vein of the low extremeties, which was compatible with autosomal dominant inheritance. CONCLUSION: FvL mutation is related to Chinese familial BCS, but is not related to Chinese sporadic BCS. FvL mutation may be a underlying pathogenicity of familial BCS. Varicose vein of the low extremeties may be one of the pathogenicity of familial BCS.

Adolescent↗
PubMed2000

[Induction of antibody to hepatitis C virus E2 antigene can be enhanced by coinjection of interleukin-2 and HCV E2 expression plasmid].

OBJECTIVE: To investigate the modulation effect of interleukin-2 (IL-2) gene on the immune response to hepatitis C virus (HCV) E2 gene when codelivered. METHODS: An expression vector of HCV E2 gene (p3E2) was constructed and its expression was detected in mammalian cells by transient expression. BALB/c mice were given injections of p3E2 only or together with IL-2 gene intraepidermally once or twice, and the induced humoral immune responses were evaluated. RESULTS: Immunohistochemistry showed that the constructed vector was successfully expressed in mammalian cells. Mice immunized with both p3E2 and IL-2 genes exhibited a significant increase in the production of anti-HCV E2 antibody compared with the mice immunized only with p3E2. CONCLUSION: Humoral immune responses of BALB/c mice to HCV E2 antigen can be enhanced when HCV E2 gene and IL-2 gene are coinjected.

Adjuvants, Immunologic↗
PubMed2000

[A report of two Chinese familial Budd-Chiari syndrome].

OBJECTIVE: To investigate the etiology of two Chinese familial Budd-Chiari syndrome (BCS). METHOD: Four patients with familial BCS (from A and B families), and the other 41 family members were detected by angiography, ultrasound Dopler, etiology analysis and Factor V Leiden (FvL) mutation analysis. RESULT: Four BCS patients were proved by angiography, 2 by ultra sound Dopler in family A. Ten members in family A were varicosis in low extremeties. FvL mutation was detected in 4 of 6 patients and 2 normal family members. A II(2), A III(7, 11, 15,) B II(10) and B III(5) had FvL mutation. The FvL mutations were compatible with Mendel hereditary law. CONCLUSION: FvL mutation may be one of main risk factors and varicosis in low extremeties may be another risk factors for familial BCS.

Adolescent↗
PubMed2000

[Relation between myocardial cAMP and renin-angiotensin system activation after acute pressure overload].

In order to investigate the relationship between cAMP and myocardial renin-angiotensin system activation after acute pressure overload, an animal model of acute pressure overload was established by constriction of abdominal aorta. It was found that angiotensin converting enzyme (ACE) mRNA and protein expression were increased markedly, ACE activity and angiotensin II content were also elevated significantly in rat heart 1 h after acute pressure overload, and the increases were kept at high level. Meanwhile the myocardial cAMP concentration was increased significantly at 0.5 h, reached the peak at day 5, and returned to normal at day 30 after operation. Then cardiomyocytes were cultured to observe whether ACE gene expression can be induced by cAMP. The results showed that elevation of cAMP content in cultured cardiomyocytes stimulated by isoproterenol (ISO, 0.01 micromol/L) increased ACE mRNA and protein expression, and also increased ACE activity and Ang II content in cultural medium. These results suggest that cAMP plays a role in the mechanism of activation of myocardial renin-angiotensin system induced by pressure overload.

Animals↗
PubMed1999

Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi.

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.

Amino Acid Sequence↗
PubMed1999

Interactions of calcineurin A, calcineurin B, and Ca2+.

Calcineurin B (CN-B) is the Ca(2+)-binding, regulatory subunit of the phosphatase calcineurin. Point mutations to Ca(2+)-binding sites in CN-B were generated to disable individual Ca(2+)-binding sites and evaluate contributions from each site to calcineurin heterodimer formation. Ca(2+)-binding properties of four CN-B mutants and wild-type CN-B were analyzed by flow dialysis confirming that each CN-B mutant binds three Ca2+ and that wild-type CN-B binds four Ca2+. Macroscopic dissociation constants indicate that N-terminal Ca(2+)-binding sites have lower affinity for Ca2+ than the C-terminal sites. Each CN-B mutant was coexpressed with the catalytic subunit of calcineurin, CN-A, to produce heterodimers with specific disruption of one Ca(2+)-binding site. Enzymes containing CN-B with a mutation in Ca(2+)-binding sites 1 or 2 have a lower ratio of CN-B to CN-A and a lower phosphatase activity than those containing wild-type CN-B or mutants in sites 3 or 4. Effects of heterodimer formation on Ca2+ binding were assessed by monitoring (45)Ca2+ exchange by flow dialysis. Enzymes containing wild-type CN-B and mutants in sites 1 and 2 exchange (45)Ca2+ slowly from two sites whereas mutants in sites 3 and 4 exchange (45)Ca2+ slowly from a single site. These data indicate that the Ca2+ bound to sites 1 and 2 is likely to vary with Ca2+ concentration and may act in dynamic modulation of enzyme function, whereas Ca(2+)-binding sites 3 and 4 are saturated at all times and that Ca2+ bound to these sites is structural.

Amino Acid Sequence↗
PubMed1999

Genetic studies on a family with acute myelogenous leukemia.

Seven cases of myelogenous leukemia--two acute erythroleukemia (AEL), four acute myelogenous leukemia (AML), and one acute myelomonocytic leukemia (AMMoL)--were found in 22 members of three consecutive generations of a family in the past 16 years (1973-1989). By using cytogenetic, hematologic, and biochemical analyses of those surviving in this family, we also found four members who might develop leukemia in the future. Southern blot analysis of one of the four members and her father (an acute leukemia patient) with a v-ERBB probe showed that the gene abnormalities consisted of a c-ERBB rearrangement (hereditary) and a rearrangement/amplification of the same gene.

Blotting, Southern↗
PubMed1999

c-kit<low Pluripotent hemopoietic stem cells form CFU-S on day 16.

Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage- (Lin-)/CD71- cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class I(high)/c-kit(low) and MHC class I(high)/c-kit<low populations from C57BL/6 Ly5.1 male mice. These two populations (1,000 cells) were transplanted into lethally irradiated (5.5 Gy x 2) C57BL/6 Ly5.2 female mice. Colony-forming unit in spleen (CFU-S) assays were carried out on days 10, 12, 14, 16, and 20. In the mice that received c-kit(low) cells, CFU-S were first detected on day 12, and the CFU-S counts gradually increased. In contrast, no visible colony was detected until day 14 in the mice that received c-kit<low cells; CFU-S were first observed on day 16. Donor-derived (Ly5.1+) cells, such as B cells, T cells, and myeloid cells, were detected by fluorescence-activated cell sorter analyses, and donor-derived erythroid cells were detected by polymerase chain reaction analyses using Y-chromosome-specific primers. Donor-derived cells in the recipients of c-kit(low) cells were detected in the spleen, bone marrow, and peripheral blood on day 12 after transplantation, while they were detected on day 16 in the mice that received c-kit<low cells. Therefore, c-kit<low cells have the capacity not only to form CFU-S on day 16 but also to reconstitute the recipients with donor-derived hematolymphoid cells 16 days after transplantation.

Animals↗
PubMed1999

A color-selectable and site-specific integrative transformation system for gene expression studies in the dematiaceous fungus Wangiella (Exophiala) dermatitidis.

To explore potential virulence factors in the dematiaceous (melanized) fungus Wangiella dermatitidis, we established a gene expression system with properties of homologous transformation and color identification. Using a polyketide synthase gene (WdPKS1) fragment for targeting, we found that 52% of transformants became albinos easily distinguishable from nonspecific transformants. Southern analysis confirmed that the integrations were at the WdPKS1 locus, which however did not affect transformant growth. With a heterologous promoter, P-glaA, enhanced expression of lacZ was found at 37 degrees C. Our results indicated that this system allows the efficient production of isogenic strains for gene function analysis in W. dermatitidis.

Amino Acid Sequence↗
PubMed1999

Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi.

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi. areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF- mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production.

Amino Acid Sequence↗
PubMed1999

Role of p53 gene mutations in human esophageal carcinogenesis: results from immunohistochemical and mutation analyses of carcinomas and nearby non-cancerous lesions.

In order to characterize p53 alterations in esophageal cancer and to study their roles in carcinogenesis, we performed gene mutation and immunohistochemical analysis on 43 surgically resected human esophageal specimens, which contain squamous cell carcinoma (SCC) and adjacent non-cancerous lesions, from a high-incidence area of Linzhou in Henan, China. A newly developed immunohisto-selective sequencing (IHSS) method was used to enrich the p53 immunostain-positive cells for mutation analysis. p53 gene mutations were detected in 30 out of 43 (70%) SCC cases. Among 29 SCC cases that were stained positive for p53 protein, 25 (86%) were found to contain p53 mutations. In five cases of SCC with homogeneous p53 staining, the same mutation was observed in samples taken from four different positions of each tumor. In a well differentiated cancer nest, p53 mutation was detected in only the peripheral p53-positive cells. In tumor areas with heterogeneous p53 staining, either the area stained positive for p53 had an additional mutation to the negatively stained area or both areas lacked any detectable p53 mutation. In the p53-positive non-cancerous lesions adjacent to cancer, p53 mutations were detected in seven out of 16 (47%) samples with basal cell hyperplasia (BCH), eight out of 12 (67%) samples with dysplasia (DYS), and six out of seven (86%) samples with carcinoma in situ (CIS). All mutations found in lesions with DYS and CIS were the same as those in the nearby SCC. In seven cases of BCH containing mutations, only three had the same mutations as the nearby SCC. The results suggest that p53 mutation is an early event in esophageal carcinogenesis occurring in most of the DYS and CIS lesions, and cells with such mutations will progress to carcinoma, whereas the role of p53 mutations in BCH is less clear.

Adult↗