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Biomedical subjects

H Y Tang

Publications and source records attributed to H Y Tang.

At least 19 recordsLinked to original sources
PubMed1997

EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

Actins↗
PubMed1996

Structural analysis of the predicted coiled-coil rod domain of the cytoplasmic bullous pemphigoid antigen (BPAG1). Empirical localization of the N-terminal globular domain-rod boundary.

The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells. BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin. All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology. To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled-coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991) J. Biol. Chem. 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified. BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration. Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing. BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled-coil formation in the rod-like tails. The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin. These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family.

Amino Acid Sequence↗
PubMed1996

Dependence of fluorodeoxyuridine-induced cytotoxicity and megabase DNA fragment formation on S phase progression in HT29 cells.

The relationship between cell cycle progression and induction of DNA double-strand breaks and cytotoxicity by exposure to fluorodeoxyuridine (FdUrd) was studied in HT29 human colon cancer cells. Fractionation of drug-treated populations by centrifugal elutriation yielded subpopulations having widely divergent abilities to progress through S phase in the presence of the drug. One of these subpopulations, which appeared to undergo coordinated growth arrest, was resistant to FdUrd cytotoxicity and DNA damage. In contrast, the subpopulation which was able to progress furthest through S phase in the presence of FdUrd underwent unbalanced growth arrest (i.e., increase in size and mass out of proportion to DNA synthesis), and displayed both DNA double-strand break formation (assayed by pulsed field gel electrophoresis) and loss of clonogenicity. When cells were elutriated prior to drug treatment, producing fractions enriched in cells at various cell cycle stages, no significant differences in sensitivity to FdUrd-induced cytotoxicity were detected among elutriation fractions. These findings support the model that, in HT29 cells, progression into and through S phase during drug treatment is an important determinant of FdUrd-induced DNA damage and cytotoxicity, but that the cell cycle position at the start of drug exposure is not a critical factor for these effects.

Antimetabolites, Antineoplastic↗
PubMed1996

Fluorodeoxyuridine-mediated cytotoxicity and radiosensitization require S phase progression.

The role of cell cycle redistribution in fluoropyrimidine-mediated radiosensitization remains unresolved. To determine if radiosensitization resulted from the redistribution of cells into a sensitive phase of the cell cycle, we assessed fluorodeoxyuridine (FdUrd)-mediated radiosensitization in flow-sorted mid-S phase HT29 human colon cancer cells. We hypothesized that if FdUrd-mediated radiosensitization were strictly the result of cell cycle redistribution, FdUrd-treated mid-S phase cells would remain as radioresistant as mid-S phase cells cultured in the absence of drug. However, we found that the mid-S phase cells from FdUrd-treated populations were markedly radiosensitized. To assess the role of S phase progression in radiosensitization, we exposed FdUrd-treated cells to aphidicolin, an inhibitor of DNA polymerase alpha, prior to irradiation. We found that aphidicolin blocked the radiosensitizing (and cytotoxic) effects of FdUrd. These results, combined with our previous observations that FdUrd-treated cells at the G1/S boundary are minimally sensitized, appear to disprove the hypothesis that sensitization results strictly from cell cycle distribution. Furthermore, they suggest that a key aspect of both FdUrd-mediated radiosensitization and cytotoxicity is S phase progression on a damaged DNA template.

Antimetabolites, Antineoplastic↗
PubMed1996

The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.

Actins↗
PubMed1995

Dependence of fluorodeoxyuridine-mediated radiosensitization on S phase progression.

Recent evidence casts doubt on the hypotheses that fluoropyrimidine-mediated radiosensitization is related to cytotoxicity or to cell cycle redistribution into the G1/S boundary. We hypothesized that cells that are capable of progressing into S phase in the presence of fluorodeoxyuridine may also be more susceptible to radiation-induced damage. To test this hypothesis, fluorodeoxyuridine (FdUrd)-treated HT29 human colon cancer cells were separated by centrifugal elutriation into four fractions (1-4) containing a range of cells from those at the G1/S boundary (fraction 1) to those which had progressed approximately 11% into S phase (fraction 4). We found that fraction 4 cells showed significantly greater radiosensitization than fraction 1 cells. We also compared the effects of fluorodeoxyuridine on HT29 and SW620 human colon cancer cells. We found that, in contrast with HT29 cells, SW620 cells arrested at the G1/S boundary and were minimally radiosensitized. Finally, we found that an increase in sensitivity was correlated with a decrease in the rate of repair of DNA double-strand and single-strand breaks (assessed by asymmetric field inversion gel electrophoresis and alkaline elution respectively). These findings are consistent with the hypothesis that fluorodeoxyuridine-mediated radiosensitization depends on S phase progression and a decreased ability to repair radiation-induced DNA damage.

Cell Separation↗
PubMed1994

Investigation on inactivated epidemic hemorrhagic fever tissue culture vaccine in humans.

From 1988 to 1992, 2479 volunteers were immunized by inactivated Meriones unguiculatus kidney cell (MUKC) vaccine. Their seroconversion rates of neutralizing antibody were about 90% after 3 doses of the vaccine. No side effects were found. The vaccine stored for 14 months at 4 degrees C still had good immunogenicity. It was shown that the vaccine was safe, effective and stable. Detailed observation was made in 75 volunteers. The results showed that the seroconversion rate by plaque reduction neutralization test in 3 doses group was higher than that in 2 doses group; but the results by reversed passive hemagglutination (RPHI), immunofluorescence assay (IFA) and ELISA were similar. Different immunization procedures (3 x 1 ml during 28 days or 3 x 1 ml during 60 days), injection routes (im or sc) and vaccine forms (containing adjuvants or not) showed no significant difference. If secondary immunization (one dose) was given one year after primary immunization, geometric mean titre of the neutralizing antibody was two fold higher than that after primary immunization.

Animals↗
PubMed1993

Resistance to fluorodeoxyuridine-induced DNA damage and cytotoxicity correlates with an elevation of deoxyuridine triphosphatase activity and failure to accumulate deoxyuridine triphosphate.

Deoxyuridine triphosphate (dUTP) misincorporation and uracil misrepair have long been implicated in fluoropyrimidine-induced DNA damage; however, the enzymatic activities responsible for these lesions have not been previously identified as critical determinants of overall sensitivity to the antitumor effects of these agents. The purpose of this study was to determine whether differences in uracil misincorporation/misrepair could account for the difference in sensitivity to fluorodeoxyuridine (FdUrd)-induced cytotoxicity and DNA damage in 2 human colorectal tumor cell lines having identical sensitivities to FdUrd-induced thymidylate synthase inhibition. Compared to HT29 cells, SW620 cells were resistant to both cytotoxicity and induction of DNA double-strand breaks, as assessed by pulse field gel electrophoresis. Alkaline elution experiments demonstrated that this resistance coincided with delayed induction of DNA single-strand breaks on parental DNA and, to a lesser extent, on nascent DNA. Following treatment with FdUrd for 24 h, HT29 cells accumulated 904 +/- 273 pmol deoxyuridine triphosphate (dUTP)/10(7) cells, whereas SW620 cells accumulated 20 +/- 7 pmol dUTP. Consistent with this difference in extent of dUTP accumulation was the observation that deoxyuridine triphosphatase levels in SW620 cellular extracts were 4.4-fold higher than in HT29 extracts. The ability to accumulate dUTP, intracellular deoxyuridine triphosphatase activity, and extent of DNA damage appear to be important determinants for predicting the response to FdUrd treatment in these cell lines.

Cell Survival↗
PubMed1993

The Pseudomonas putida ML2 plasmid-encoded genes for benzene dioxygenase are unusual in codon usage and low in G+C content.

Benzene dioxygenase, catalyzing the oxidation of benzene to cis-1,2-dihydroxy-cyclohexa-3,5-diene, comprises four polypeptides that are encoded by plasmid pHMT112 of Pseudomonas putida ML2. In this study, the nucleotide (nt) sequences of four genes encoding this enzyme (bedC1C2BA) were determined, and the amino acid (aa) sequences were deduced. The sequence showed significant homology with the chromosomally encoded benzene dioxygenase and toluene dioxygenase genes (73-77% for nt and 83-99% for aa), but not the plasmid-encoded naphthalene dioxygenase genes (20-26% for nt and 32-36% for aa). A conserved motif (Cys-Xaa-His-15-to-17 aa-Cys-Xaa2-His, where Xaa is any aa), proposed to bind the Rieske-type [2Fe-2S] cluster, was identified in the deduced aa sequence of the iron-sulfur proteins. Three regions were also identified in the flavoprotein which are likely to be involved in FAD and NAD+ binding. The gene order of bedC1C2BA is consistent with most ring-hydroxylating dioxygenases isolated from Pseudomonas. However, the G+C content of 47% is in contrast to the high G+C content of the Pseudomonas chromosome (63%) and other Pseudomonas plasmids (57%), and with its unique codon usage preference this suggests that bedC1C2BA originated from a host derived from a different genus.

Amino Acid Sequence↗
PubMed1992

Variations in patterns of DNA damage induced in human colorectal tumor cells by 5-fluorodeoxyuridine: implications for mechanisms of resistance and cytotoxicity.

We have previously shown that treatment of the HT29 human colorectal tumor (HCT) cell line with 100 nM 5-fluorodeoxyuridine (FdUrd) induces DNA fragments ranging from 50 kilobases to 5 megabases. The studies reported here were conducted to characterize the kinetics, concentration dependence, and pharmacologic specificity of this process and to determine if such fragmentation varies among HCT cell lines. HT29 and SW620 cells yielded similar fragment size distributions upon treatment with either FdUrd or CB3717 [a folate analog inhibitor of thymidylate synthase (TS)]. With either of these agents the SW620 line required higher drug concentrations or longer incubation times than HT29 cells to achieve a given level of fragmentation or cytotoxicity, even though the two cell lines are equally sensitive to FdUrd-induced TS inhibition. These data indicate that SW620 resistance is not due to a lesion in the events leading up to TS inhibition but it may be due to a difference in the steps following TS inhibition. Aphidicolin, a DNA polymerase inhibitor, did not cause substantial fragmentation or cytotoxicity in these two cell lines, demonstrating that the fragmentation response to the other two drugs is not a general consequence of DNA synthesis inhibition. A third HCT line, HuTu80, gave rise only to a smaller and more discrete population of DNA fragments, ranging from approximately 50 to 200 kilobases, following exposure to FdUrd. Similar patterns were seen in this line upon treatment with CB3717 or aphidicolin, indicating that this fragmentation pattern is not specific to TS inhibition and may be characteristic of a more general response than that seen in the other two cell lines. DNA fragments induced by FdUrd in HuTu80 cells did not degrade into smaller pieces, demonstrating that the process by which they are formed is distinct from apoptosis. We conclude that the responses of HCT cells to FdUrd-induced TS inhibition vary significantly, that these differences may reflect heterogeneity in the mechanism of DNA damage formation, and that, in some cases, FdUrd resistance may be due to alterations in the fragmentation process.

Cell Survival↗
PubMed1992

Type I keratinocyte transglutaminase: expression in human skin and psoriasis.

A 92-kD transglutaminase (TGase K), expressed in human cultured keratinocytes and stratum corneum, catalyzes a critical step in the formation of the cornified envelope of terminal differentiation. A rabbit polyclonal antibody to TGase K was used to isolate overlapping cDNA clones from a human keratinocyte cDNA expression library. The cDNA clones were sequenced and unequivocally identified as TGase K by comparison to the N-terminal amino acid sequences of two cyanogen bromide fragments from the purified enzyme. The mRNA for Tgase K is expressed in cultured keratinocytes but not in A431 squamous carcinoma cells, in fibroblasts, or in other non-epithelial tissues and cells. Although TGase K protein expression is limited to the upper layers of normal epidermis, the mRNA is generally present throughout the epidermis, suggesting the possibility of post-transcriptional regulation. Precocious expression of TGase K protein occurs in psoriasis, and quantitative Northern blot analysis of TGase K mRNA from normal and involved epidermal biopsies from psoriasis patients suggests that TGase K mRNA levels are increased in psoriatic lesions. By using quantitative laser scanning confocal microscopy (LSCM) and in situ hybridization, the increase of the TGase K mRNA was in the range of 3-7 times in the psoriatic epidermis and was significantly higher compared with normal skin and with paired adjacent skin. Quantitative LSCM provides a powerful and direct method for analysis of gene expression in skin.

Amino Acid Sequence↗
PubMed1992

[A study on the location of immuno-suppressive factor(s) in restraint rats and mice].

A serum lymphocyte-proliferation suppressive factor(s) induced by restraint stress over 10 h was found in previous studies in both rats and mice. The present study was undertaken to investigate the sites of its production. The results show that large doses of irradiation and cyclophosphamide (CY) decreased the total number of splenic nucleated cells, but the production of the suppressive factor was inhibited only by irradiation. This indicates that the drop in total number of lymphocytes does not play any key role in the production of the serum suppressive factor. Cell classification showed that the ratio of T to B cell was decreased by radiation but increased by CY, suggesting that this ratio may be relevant to the production of the factor. Inhibition of the production was also observed in nude mouse (an animal showing a lack of T cell activity), again supporting that T cells are involved in the production of the inhibitory factor.

Animals↗
PubMed1991

IL-4-dependent IgE switch in membrane IgA-positive human B cells.

IgE responses by human B cells, separated according to membrane Ig classes, were analyzed in a clonal assay using EL-4 thymoma cells as helper cells, T cell supernatant, and rIL-4. In cultures seeded by means of the autoclone apparatus of the FACS, IgE responses were generated frequently by either IgM (mu+/gamma-alpha-) or IgA (alpha +/mu-)-positive B cells (16 and 14% of the Ig producing wells, respectively), but rarely by IgG (gamma +/mu-)-positive B cells (1.3% of Ig producing wells). The total amounts of Ig secreted by IgM-, IgG-, or IgA-positive cells and the total proportions of responding autoclone wells (23-27%) were comparable. All IgE secretion was IL-4 dependent. When the Ig secretion patterns from alpha +/mu- vs alpha +/mu-epsilon- B cells were compared, most autoclone wells from both types of cells produced IgA only, and similar proportions of IgA producing wells (6.2 and 6.0%) also secreted IgE. In addition, IgE restricted responses occurred 6 times more frequently with alpha +/mu- than with alpha +/mu-epsilon- cells, which suggests that membrane IgA+E double-positive, IgE committed B cells occur in vivo. The isotype pattern generated by alpha +/mu-epsilon- B cells cannot be explained by a chance assortment of separate IgA and IgE precursors or by cytophilic antibody. Thus, IL-4 dependent switch to IgE occurred frequently in IgM- or IgA-positive, but rarely among total IgG-positive, B cells. This could be relevant to IgE production in mucosal tissues rich in IgA expressing B cells.

B-Lymphocyte Subsets↗
PubMed1990

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90.

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.

Animals↗
PubMed1989

Efficacy of inactivated vaccine containing cyto-hemagglutinin against epidemic hemorrhagic fever in rabbits.

Using the Z-10 strain of epidemic hemorrhagic fever virus (EHFV) as seed, and the primary cell of Meriones unguiculatus kidney tissue as incubation cell, a propiolactone inactivated epidemic hemorrhagic fever (EHF) vaccine was prepared, according to a similar procedure required for the production of biological products such as the Japanese B encephalitis vaccine. Besides the EHFV antigen detected by ELISA or reversed passive hemagglutination test (RPHA) as were used for the formalin inactivated vaccine, higher titres (1:128-1:1024) of EHFV hemagglutinin antigen was also detected in this EHFV vaccine. Immunization with twice intramuscular injection of this vaccine produced high titred (1:20-1:160) neutralizing antibody and low titred (1:10-1:20) hemagglutination antibody, in addition to the immunofluorescence (IF) and reversed passive hemagglutination inhibition (RPHI) antibodies. These results indicated an apparent difference in the immunogenicity between the beta-propiolactone and formalin inactivated EHF vaccines. With the approval of the Ministry of Health, human test is now underway in this laboratory.

Animals↗
PubMed1987

Recent advances in microlymphatic surgery in China.

Development and advances in clinical and research work on limb lymphedema in China has progressed rapidly in recent years. The authors first performed successful lymphaticovenous anastomosis using the operating microscope for limb lymphedema in China in May, 1979. By 1983, surgery on 48 lymphedematous limbs in the authors' clinic had given good results in one-third of the cases. By the same year, 185 limbs with lymphedema were treated by lymphaticovenous anastomosis throughout China with excellent results achieved in 72.9% of the cases. Lymphology was investigated using animal models, intraluminal pressure, and venous graft substitution of lymphatics.

Animals↗