Biomedical subjects

M Fang

Publications and source records attributed to M Fang.

At least 73 records · Page 4Linked to original sources

PubMed1998

Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Amino Acid Sequence↗

PubMed1998

Intracellular calcium distribution in apoptosis of HL-60 cells induced by harringtonine: intranuclear accumulation and regionalization.

Harringtonine (HT), an anticancer drug with high chemotherapeutic efficiency to human chronic granulocytic/myelomonocytic leukemia, has been reported to rapidly induce apoptosis in HL-60 cells in a wide scope/range of dosage by investigators from our lab and others. In the present studies, by using video enhancement contrast (VEC) microscopy, we dynamically analyzed changes in intracellular calcium distribution in a single HL-60 cell over the period from the initiation of apoptosis to the obvious appearance of chromatin condensation. The results from this paper demonstrated the striking distinction of intracellular calcium distribution at different time points after treatment with HT. Before treatment in normal HL-60 cells the highest [Ca2+]i accumulation was observed in the peri-nuclear area and the lowest was observed in the nucleus; after treatment with 1 microg/ml HT for 30 min intracellular calcium diffused all over the cell compartments, while intranuclear calcium increased comparatively and significantly. The phenomenon of intranuclear calcium accumulation was further confirmed by using laser scanning confocal microscopy (LSCM). In addition, co-localization of the highest calcium region with condensed chromatin in apoptotic HL-60 cells was also observed by LSCM. Our results suggest that two sequential alterations of intracellular calcium distribution occurred in apoptotic HL-60 cells induced by HT, i.e. (a) accumulation of calcium in the nucleus and (b) regionalization in a specific nuclear region.

Antineoplastic Agents↗

PubMed1998

A promoter relay mechanism for sequential gene activation.

The effect of DNA supercoiling on gene expression is dependent not only on specific genes but also on the sequence context of the genes. This position-dependent supercoiling effect on gene activation is best illustrated in the study of the suppression of the leu-500 mutation of the leuABCD operon in a Salmonella typhimurium topA mutant. In this communication, we report a novel promoter relay mechanism whereby several genes are sequentially expressed in a position-dependent manner: the ilvIH promoter (pilvIH) activates a cryptic leuO promoter (pleuO) located between the two divergently arrayed ilvIH and leu-500 promoters. Both the cis-acting pleuO activity and the trans-acting LeuO protein are necessary for subsequent activation of the leu-500 promoter (pleu-500). Furthermore, pleuO can be functionally replaced with the inducible tac promoter (ptac) for leu-500 activation, suggesting that transcription-driven DNA supercoiling underlies the relay mechanism. This is the first example of several related genes communicating via a promoter relay mechanism for their coordinated expression.

Amino Acid Sequence↗

PubMed1997

GFRalpha-2 and GFRalpha-3 are two new receptors for ligands of the GDNF family.

The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFRalpha-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFRalpha-1 or GFRalpha-2, a novel receptor related to GFRalpha-1. Both GFRalpha-1 and GFRalpha-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFRalpha-1 or GFRalpha-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFRalpha-1 bind GDNF more efficiently than neurturin, while cells expressing GFRalpha-2 bind neurturin preferentially. Cross-linking and Ret activation data also suggest that while there is cross-talk, GFRalpha-1 is the primary receptor for GDNF and GFRalpha-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFRalpha receptor family. This putative receptor, designated GFRalpha-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFRalpha-1 and GFRalpha-2. Analysis of the tissue distribution of GFRalpha-1, GFRalpha-2, GFRalpha-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFRalphas and Ret are expressed in many of the same tissues, suggesting that GFRalphas mediate the action of GDNF family ligands in vivo.

Amino Acid Sequence↗

PubMed1997

Enhanced Reverse Transcriptase Polymerase Chain Reaction for Prostate-specific Antigen Combined With Needle Biopsy Results: A Superior Predictor of pT3 Disease.

Background: Preoperative staging for prostate cancer underestimates the final pathology stage in approximately 40-50% of the cases. Previous work from our institution demonstrated that an enhanced reverse transcriptase polymerase chain reaction (RT-PCR) assay for prostate-specific antigen (PSA) enabled more accurate staging of presumably localized prostate cancer. The goal of the current study is to determine if needle biopsy results when combined with the RT-PCR for PSA assay are a better predictor of final pathology stage. Methods and Results: One hundred sixty-two men with needle biopsy-diagnosed prostate cancer had blood drawn for the RT-PCR for PSA assay before undergoing radical prostatectomy. Polymerase chain reaction primers specific for the PSA gene were run, along with appropriate controls. Tumor was characterized using the TMN staging system: organ confined (pT2a-c), capsular penetration (pT2a-b), seminal vesicle involvement (pT3c). Surgical margins and lymph nodes were also evaluated. Of the 162 patients, the majority had localized disease by digital rectal examination: T2 = 97%, and T3 = 3%. On needle biopsy, 48 cases (30%) had a Gleason score >/=7 and 35 cases (22%) had perineural involvement (PNI). The RT-PCR for PSA assay was positive in 50 patients (31%). Final pathology revealed 39% of patients had pT3 disease; none of the 162 patients had lymph node involvement. Statistical analysis revealed that a Gleason score >/=7 had 81% specificity and 46% sensitivity in predicting pT3 disease (odds ratio 3.6). The presence of PNI on needle biopsy was 89% specific and 38% sensitive in predicting pT3 disease (odds ratio, 4.9). The RT-PCR for PSA assay was 89% specific and 62% sensitive in predicting pT3 disease (odds ratio, 13.0). All 14 cases with both RT-PCR for PSA and PNI positivity had pT3 disease. Logistic regression analysis demonstrated the independent predictive strength of PNI on needle biopsy, Gleason score >/=7, and RT-PCR for PSA positivity for identifying pT3 disease; their combined odds ratio was more than 180. Conclusions: Using the RT-PCR for PSA assay in conjunction with needle biopsy results increases the predictive strength for pT3 disease in patients with presumed organ-confined prostate carcinoma.

Journal Article↗

PubMed1997

Cyclosporine inhibited calcium-mediated apoptosis of HL-60 cells.

AIM: To study the effects of cyclosporine (Cyc) on apoptosis of HL-60 cells. METHODS: Apoptotic cells induced by harringtonine (Har), camptothecin (Cam), or calcimycin (Cal), thapsigargin (Tha) were identified with DNA electrophoresis, morphology, and flow cytometry. Relative [Ca2+]i alteration of apoptotic HL-60 cells were determined with flow cytometry. RESULTS: Cal 1 mg.L-1 or Tha 0.5 mg.L-1 induced apoptosis of HL-60 cells. This effect was inhibited by nontoxic concentration of Cyc 1 mg.L-1. Cyc did not inhibit Har- or Cam-induced apoptosis of HL-60 cells. Both Cal and Tha increased intracellular calcium, whereas Har or Cam did not. CONCLUSION: Cyc inhibited apoptosis only induced by calcium increasement in HL-60 cells. The mechanism of apoptosis induced by Cal or Tha was different from that by Har or Cam.

Anti-Bacterial Agents↗

PubMed1997

[Sequence of HLA-DQA1 promoter region in the Han people].

Polymorphism of HLA-DQA1 promoter region (QAP) in the Han people has been identified. The results revealed a number of differences, some of which are in the critical class II boxes, and generally conserved in HLA-DQA1 promoter region. The major differences occurred in the X box, Y box and S box. Within the X box, the Hans carry a A at position -111, instead of a G, and a G or a A can be present at position -98. Within the S box, the Hans carry a G at position -131. Within the Y box, position -71 is a A rather than a G. Some single base substitutions have been detected from IDDM patients at the 5'-flanking region of the S box and between X box and Y box. Particularly, the insertion of CCA bases has been identified at the position between -157 and -158 in a IDDM patient. These data suggest that the polymorphism of HLA-DQA1 promoter region may play a role in susceptibility to IDDM.

Adult↗

PubMed1997

[Detection of flaviviruses by reverse-transcription polymerase chain reaction with universal primer set].

For the purpose of early and rapid diagnosis of flavivirus infection, the universal primer set was selected on the NS1 gene. Another five different internal primers were selected on the NS1 genes of DEN1, 2, 3, 4 and JEV. With the universal primer set designed, the NS1 fragment about the size of 413 bp and with the five internal primers, the NS1 fragments about the sizes of 262 bp (DEN1), 189 bp (DEN2), 392 bp (DEN3), 97 bp (DEN4) and 323 bp (JEV) were amplified respectively. The reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify gene fragments of flaviviruses. cDNA of DEN1, 2, 3, 4 and JEV were successfully amplified with universal primer set and internal primers. Sera from 78 patients with dengue fever were assayed by nested PCR, DEN1 was detected from 18 of the 40 patients' sera and DEN2 was detected from 48 patients' sera. Sera from 42 patients with Japanese encephalitis were also assayed by nested PCR, JEV was detected from 35 of the 42 patients' sera. By nested PCR, we completed identification of flaviviruses within 2 days. The results showed that this method has the potential value in rapid clinical diagnosis of flavivirus infection.

DNA Primers↗

PubMed1996

Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.

Alleles↗

PubMed1996

GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

Amino Acid Sequence↗

PubMed1996

Alteration of the pharmacokinetics of small proteins by iodination.

The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.

Animals↗

PubMed1996

The yeast BSD2-1 mutation influences both the requirement for phosphatidylinositol transfer protein function and derepression of phospholipid biosynthetic gene expression in yeast.

The BSD2-1 allele renders Saccharomyces cerevisiae independent of its normally essential requirement for phosphatidylinositol transfer protein (Sec14p) in the stimulation of Golgi secretory function and cell viability. We now report that BSD2-1 yeast mutants also exhibit yet another phenotype, an inositol auxotrophy. We demonstrate that the basis for this Ino- phenotype is the inability of BSD2-1 strains to derepress transcription of INO1, the structural gene for the enzyme that catalyzes the committed step in de novo inositol biosynthesis in yeast. This constitutive repression of INO1 expression is mediated through specific inactivation of Ino2p, a factor required for trans-activation of INO1 transcription, and we show that these transcriptional regulatory defects can be uncoupled from the "bypass Sec14p" phenotype of BSD2-1 strains. Finally, we present evidence that newly synthesized phosphatidylinositol is subject to accelerated turnover in BSD2-1 mutants and that prevention of this accelerated phosphatidyl-inositol turnover in turn negates suppression of Sec14p defects by BSD2-1. We propose that, in BSD2-1 strains, a product(s) generated by phosphatidylinositol turnover coordinately modulates the activities of both the Sec14p/Golgi pathway and the pathway through which transcription of phospholipid biosynthetic genes is derepressed.

Carrier Proteins↗

PubMed1996

[Induction of expression of MDR 1 gene by retinoic acid and DMSO and effects on rhodamine-123 efflux in HL-60 cell lines and resistant sublines].

Using dot blot hybridization and flowcytometry, the effects of differentiation inducers retinoic acid (RA) and dimethyl sulfoxide (DMSO) on the resistant level of HL-60 cells and its resistant subline cells were studied. When the cells were treated with RA 1 mumol.L-1 for 24 h, the expression of MDR 1 mRNA evidently increased in both HL-60 and its multidrug resistant subline cells. The efflux of Rho-123 in the multidrug resistant subline cells was slightly decreased. But, when the cells were treated with 2% DMSO for 24 h the efflux of Rho-123 increased obviously. The results suggest that RA can induce the expression of MDR1 gene but perhaps inhibit the function of pump glycoprotein 170 (Pgp-170) through phosphorylation/dephosphorylation pathway. However, DMSO could induce the expression of full function of Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

[Protein kinase inhibitor staurosporine enhances cytotoxicity of antitumor drugs to cancer cells].

Treated with low dosage (5 ng.ml-1) of staurosporine for 18 h, human embryo lung 2BS cells were blocked at the G1/S boundary, but human gastric carcinoma BGC-823 cells still kept their cell cycle. In comparison with IC50 of 2BS and BGC-823 cells treated with cell cycle phase specific antitumor drugs adriamycin, Ara-C and BLM A5 alone or combined with staurosporine 5 ng.ml-1, the IC50 values increased from 0.325 microgram.ml-1, 5 micrograms.ml-1 and 6.5 micrograms.ml-1 to 0.45 microgram.ml-1, 10 micrograms.ml-1 and 6.5 micrograms.ml-1, respectively in 2BS cells; but decreased from 0.325 microgram.ml-1, 25 micrograms.ml-1 and 1.1 micrograms.ml-1 to 0.07 microgram.ml-1, 6.25 micrograms.ml-1 and 0.4 microgram.ml-1, respectively in BGC-823 cells. These results suggest that combination of staurosporine 5 ng.ml-1 with antitumor drugs showed different effects on tumor cells and normal cells. With the GSH fluorescent probe mBCL, we found that GSH contents increased in 2BS cells treated with staurosporine 5 ng.ml-1.

Antibiotics, Antineoplastic↗

PubMed1996

[Changes of intracellular calcium, calmodulin in normal and tumor cells traggered by staurosporine].

Treated with low dosage staurosporine, the normal cell 2 BS were arrested in G 1 phase, but tumor cell BGC-823 were not. We measured the intracellular calcium, calmodulin, Ca(2+)-activited calmodulin (Ca(2+)-CaM) contents of single cells according to cell cycle with microphotometry. Our results showed that treatment of 5 ng/ml staurosporine for 18 h caused the changes of calcium and calmodulin. In 2 BS cells, CaM leval decreased in G 1 and S phase. In each cell cycle phase of BGC-823 cells, there was no changes in CaM level, but Ca(2+)-CaM level increased. The causes of that staurosporine blocked 2 BS cells at G 1 phase but not effected on cell cycle progression in BGC-823 cells may be the results of staurosporine decreased the contents of CaM at G 1 phase and inhibited the phosphoralation of p107 in 2 BS cells.

Calcium↗

PubMed1996

[Location of the surface protein antigen I/II on Mutans Streptococci with immunogold electron microscope].

Mutans Streptococci possess a number of surface protein antigens. The surface protein antigen I/II with a molecular mass of 190,000 is considered to play an important role in the initial attachment to tooth surface. The antigen is highly immunogenic and has been successfully used as a vaccine against dental caries. The object of this study is to locate the surface protein antigen I/II of Mutans Streptococci with immunogold electron microscope. The results suggest that (1) antigen I/II locate on the cell wall surface of serotype c, e, f; (2) antigen I/II locate on the "fuzz coat" of the cell wall of serotype d, g; (3) some antigen I/II locate at the surface of cell wall of serotype a; (4) antigen I/II are absent on the cell wall surface of serotype b strain.

Antigens, Surface↗

PubMed1996

[Apoptosis of HL-60 cells induced by Harringtonine: membrane blebs, nucleus blebs and chromatin condensation].

Using Video Enhancement Contrast (VEC) microscopy, we recorded the morphological changes of same HL-60 cell in the processes of apoptosis induced by harringtonine. Our results show that all of apoptotic cells need several nucleus blebs before their chromatin condensation. Every nucleus bleb is induced by a relative membrane bleb. The number of membrane blebs is much higher than that of nucleus blebs, so there are only some of membrane blebs which can induce nucleus blebs. It suggested that membrane and nucleus blebs probably are related to apoptotic chromatin condensation. After HL-60 cells pretreated with cytochalasin B(CB), apoptotic chromatin condensation delayed eight hours, but no membrane bleb, nucleus bleb and apoptotic body formed eventually. So membrane and nucleus blebs during apoptosis are related to microfilament re-organization and can accelerate apoptotic chromatin condensation, but are unnecessary for apoptotic chromatin condensation. All this suggested that nuclear changes and cytoplasmic changes during HL-60 cell apoptosis are independent.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Correlation between expression of mdr-1 gene and oncogenes in human promyelocytic leukemic HL60 cell line and sublines.

AIM: To study the relationship between expression of oncogenes and multiple drug resistant (MDR) phenotype. METHODS: The drug resistant level of HL60 cell line and its sublines were determined with flow cytometry. RNA Dot blot hybridization was used to identify the expression of oncogenes and mdr-1 gene. RESULTS: The expression of mdr-1 gene was in the opposite relation with c-myc expression, but in the positive relation with c-H-ras gene expression in the multiple drug resistant cell lines. In non-MDR cell line HL60/RA, the expression levels of mdr-1, c-myc, and c-H-ras were the same as HL60 parental cells. CONCLUSION: Multiple drug resistance is related to not only mdr-1 expression, but also some oncogenes expression level.

Drug Resistance, Multiple↗