Biomedical subjects

J Cao

Publications and source records attributed to J Cao.

At least 181 records · Page 10Linked to original sources

PubMed1999

[Detection of human papillomavirus gene in biopsies from colon carcinoma by PCR].

OBJECTIVE: To detect the human papillomavirus gene (HPVs) in biopsies from colon carcinoma by PCR and to study the relationship between HPV infection and colon carcinoma. METHODS: 46 biopsies from colon carcinoma, 10 biopsies from tissue around carcinoma and 16 control biopsies were collected and DNA were extracted. The HPV DNA in biopsies were amplified by PCR using general primers (GP) and primers for HPV6/11, HPV16/33 and HPV18. RESULTS: 20 out of 46 biopsies from colon carcinoma showed HPV gene positive, HPV6/11, HPV6/33 and HPV18 positivity were 3, 11 and 6 respectively. HPV DNA was found in 1 case from 10 biopsies of surrounding tissue of carcinoma. 16 control biopsies were negative. CONCLUSION: HPV may be related to the etiology of colon carcinoma.

Colorectal Neoplasms↗

PubMed1998

The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A.

Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.

Enzyme Activation↗

PubMed1998

Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor.

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.

Amino Acid Sequence↗

PubMed1998

In situ detection of frameshift mutations in mouse cells.

A reporter gene system that allows in situ detection of cells that have suffered a specific frameshift mutation was developed. To construct the reporter gene, the open reading frame of a human placental alkaline phosphatase (PLAP) gene was disrupted by insertion of either 5 or 7 G:C basepairs, which formed mutant alleles carrying 9 and 11 consecutive G residues, respectively. The mutant PLAP genes did not produce alkaline phosphatase activity in cultured mouse cells in transient transfection assays. Several cell lines that contained integrated copies of the mutant PLAP genes were made. Histochemical staining of fixed cells showed that these cell lines contained a small number of cells that expressed PLAP activity and bound antibodies directed against PLAP. Cells carrying the allele with 11 consecutive G residues (G11 allele) acquired PLAP activity at a rate between 2 x 10(-3) and 2 x 10(-4) events per cell per generation, depending on the cell line. Cells carrying the allele with 9 consecutive G residues (G09 allele) acquired PLAP activity at a rate between 2 x 10(-5) and 2 x 10(-6) events per cell per generation, depending on the cell line. Cultures of PLAP+ cells were derived from cell lines carrying PLAP mutant genes. All the cells in these cultures had PLAP activity and bound anti-PLAP antibody. PLAP mRNA levels were the same in cultures where all cells were PLAP+ and in cultures where less than 1% of the cells expressed PLAP activity. DNA sequence analysis of PLAP+ cells showed that the G11 allele reverted by losing one basepair, and the G09 allele reverted by gaining one basepair.

Alkaline Phosphatase↗

PubMed1998

Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline.

Expression of HIV-1 Vpr causes cell cycle G2 arrest, change in cell shape, and cell death over a large evolutionary distance ranging from human to yeast cells. As a step toward understanding these highly conserved Vpr functions, we have examined the effect of Vpr on cytoskeletal elements and the viability of fission yeast. We demonstrate that the changes in cell morphology induced by Vpr in fission yeast are caused by several underlying cellular abnormalities, including increased biosynthesis of chitin in the cell wall, disruption of the actin cytoskeleton, and altered polarity for cell growth. The extent of these cellular alterations and cell survival correlates with the level of vpr expression. Accompanying cell death, Vpr induces aberrant nuclear morphologies in fission yeast which are similar to those found during the apoptosis induced by Vpr in mammalian cells. The Vpr-induced cytopathic effects and cell death can be suppressed by treatment with pentoxifylline, a compound that inhibits HIV-1 viral replication and suppresses Vpr-induced cell cycle G2 arrest in human and fission yeast cells. The results presented here suggest that pentoxifylline suppresses the effects of Vpr by blocking interactions of Vpr with cellular proteins. Given that pentoxifylline has potential therapeutic value in blocking the effects of Vpr in HIV-infected patients, understanding the molecular mechanisms by which pentoxifylline antagonizes Vpr may have general implications for HIV therapy.

Anti-HIV Agents↗

PubMed1998

In vivo phosphorylation of phosphofructokinase from the bivalve mollusk Mytilus galloprovincialis.

The phosphorylation state of phosphofructokinase from the mantle tissue of the facultative anaerobe mollusk Mytilus galloprovincialis was determined by a back-phosphorylation technique. The incubation of intact mantle tissue with 8-bromoadenosine 3':5'-cyclic monophosphate increased significantly the phosphate content of phosphofructokinase, which indicates that the enzyme can be phosphorylated in vivo by endogenous cAMP-dependent protein kinase. The phosphate content of mussel phosphofructokinase changes significantly during the year, in agreement with the kinetic data that show a more active enzyme form in earlier autumn. These results suggest that cAMP-dependent phosphorylation of phosphofructokinase can be partially responsible for the observed glycolytic changes associated with the annual gametogenic cycle that takes place in the mantle tissue of the mollusk. On the contrary, no differences were observed between aerobic and 24-h hypoxic mussels with regard to the phosphorylation state and the kinetic constants of phosphofructokinase. This result is inconsistent with the hypothesis that phosphorylation of phosphofructokinase is involved in the glycolytic depression that takes place during the long-term environmental hypoxia that the mollusk can undergo.

Animals↗

PubMed1998

Reduced fertility in female mice lacking copper-zinc superoxide dismutase.

Copper-zinc superoxide dismutase (CuZn-SOD) is believed to play a major role in the first line of antioxidant defense by catalyzing the dismutation of superoxide anion radicals to form hydrogen peroxide and molecular oxygen. Recent studies have shown that missense mutations in this gene contribute, evidently through a gain-of-function mechanism, to about 20% of familial amyotrophic lateral sclerosis. To define further the physiologic role of this enzyme, a model of mice deficient in this enzyme was generated using gene targeting technology. Mice lacking this enzyme were apparently healthy and displayed no increased sensitivity to hyperoxia. However, they exhibited a pronounced susceptibility to paraquat toxicity. Most surprisingly, female homozygous knock-out mice showed a markedly reduced fertility compared with that of wild-type and heterozygous knock-out mice. Further studies revealed that although these mice ovulated and conceived normally, they exhibited a marked increase in embryonic lethality. These data, for the first time, suggest a role of oxygen free radicals in causing abnormality of female reproduction in mammals.

Animals↗

PubMed1998

Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP).

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2.MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

Amino Acid Sequence↗

PubMed1998

Choriocapillaris degeneration and related pathologic changes in human diabetic eyes.

OBJECTIVES: To measure the extent of choriocapillaris degeneration (CCD) in diabetic choroids and to study the association of CCD with choroidal neovascularization and pathologic changes in Bruch's membrane-like basal laminar deposits. MATERIALS AND METHODS: Human choroids from 10 postmortem subjects (diabetic, 5 [group 1]; nondiabetic, 5 [group 2]) were incubated for the histochemical demonstration of alkaline phosphatase and nonspecific esterase activities, permitting analysis of the choroidal vasculature and polymorphonuclear leukocytes, respectively. The tissue was then flat embedded and sectioned for structural analysis. Areas of CCD were measured in the flat perspective by computer-assisted image analysis and verified in cross-sections of flat-embedded tissue. RESULTS: The CCD in choroids from subjects with diabetes (group 1) appeared in 2 patterns: diffuse (partial loss of alkaline phosphatase activity in a poorly defined area, ie, degeneration of some capillary segments) and focal (complete degeneration of choriocapillaris or loss of alkaline phosphatase activity in a relatively well-defined area). The mean+/-SD percentage of the choroid with focal CCD in group 1 was 5.08%+/-1.13% of the total choroidal area vs 1. 16%+/-0.35% in group 2 (P<.001). Focal CCD in group 1 was more prominent in the posterior pole than in the peripheral choroid. Choroidal neovascularization was associated with some areas of diffuse CCD in group 1. Pathologic changes in Bruch's membrane-like basal laminar deposits were often associated with CCD; the thickness of the deposits was greater in group 1 than in group 2 and greater in areas with focal CCD than in areas with diffuse or no CCD. CONCLUSION: The percentage of choroid with focal CCD in group 1 choroids was more than 4-fold greater than that in nondiabetic choroids. The presence of CCD was related to basal laminar deposits and, in some cases, to choroidal neovascularization.

Aged↗

PubMed1998

Use of human placental alkaline phosphatase transgenes to detect somatic mutation in mice in situ.

Methods for in situ detection of cells that have suffered a specific mutation would be valuable for understanding somatic genetic mosaicism, a phenomenon that underlies a variety of diseases including cancer. Such methods would also be valuable in studying changes in gene expression, whether programmed by the cells or caused by exogenous forces, such as exposure to genotoxins or infection by a virus. To improve methods for detection of genetic change at the cellular level in animal tissues, we used the human placental alkaline phosphatase (PLAP) gene. The PLAP gene sequence was modified such that it could no longer produce functional PLAP enzyme. Mutant PLAP genes were placed in the mouse genome, and populations of cells carrying these mutant PLAP genes were studied to determine the fraction of cells that would acquire PLAP activity. Spontaneous and induced reversion of mutant PLAP genes was studied in cultured cells and in the tissues of transgenic mice. The data obtained from these studies show the utility of in situ reporter genes such as PLAP for detection of variant cells within a tissue.

3T3 Cells↗

PubMed1998

In vitro side-view imaging technique and analysis of human T-leukemic cell adhesion to ICAM-1 in shear flow.

The objective of the present study is to apply a novel side-view imaging technique to investigate T-leukemic Jurkat cell adhesion to a surface-immobilized ICAM-1 in shear flow, a ligand for leukocyte LFA-1. Images have revealed that Jurkat cell adhesion on ICAM-1 under flow conditions in vitro is quasistatic. The cell-substrate contact length steadily increased with time during the initial cell attachment to the ICAM-1-coated surface and subsequently decreased with time as the trailing edge of the cell membrane peeled away from the substrate under the influence of fluid shear forces. Changes in flow shear stresses, cell deformability, or substrate ligand strength resulted in a significant change in the characteristic adhesion binding time and contact length. A 3-D flow field with shear stresses acting on an adherent cell was calculated by using finite element methods based on cell shapes obtained from the in vitro images. The maximum shear stress acting on an actual cell body was found to be 3-5 times greater than the upstream inlet wall shear stress and was influenced by the extent of cell deformation within the flow channel. Therefore, the application of such a side-view imaging technique has provided a practical assay to study the mechanics of cell-surface adhesion in 3-D. The elongation of cells in shear flow tempers hydrodynamic shear forces on the cell, which affects the transients in cell-surface adhesion.

Biomechanical Phenomena↗

PubMed1998

A method for the purification of cAMP-dependent protein kinase using immunoaffinity chromatography.

A rapid and efficient method for purifying cAMP-dependent protein kinase (PKA) holoenzyme based on immunoaffinity chromatography was developed. The affinity column was prepared by coupling a polyclonal antibody raised against the PKA regulatory subunit to NHS-activated Sepharose. The holoenzyme purified by this procedure from the bivalve mollusk Mytilus galloprovincialis was shown to be fully active as judged by (1) its cAMP-binding activity, (2) its cAMP-dependent protein kinase activity, and (3) its autophosphorylation ability. Moreover, together with both regulatory and catalytic subunits, which constitute the PKA holoenzyme, a protein with a molecular mass of approximately 200 kDa was copurified, and results from gel-filtration chromatography showed that it was associated with a fraction of PKA. Therefore, this immunoaffinity purification technique could also be useful to isolate such proteins as interact with PKA in vivo.

Animals↗

PubMed1998

LEA-1/ICAM-1 interaction is essentially involved in the pathogenesis of delayed-type hypersensitivity-induced liver injury to picryl chloride.

The kinetics of lymphocyte function associated antigen 1 (LFA-1) expression on spleen cells (SPC) and liver non-parenchymal cells (NPC), and intercellular adhesion molecule 1 (ICAM-1) expression on hepatocytes (HC) was examined in acute liver injury mice induced by a DTH reaction to picryl chloride (PCl). The peak expression of LFA-1 on SPC was seen at 6 hr after eliciting liver injury, and then that of LFA-1 on NPC and ICAM-1 on HC appeared at 12 hr. Thereafter, the serum ALT elevation reached to a peak at 18 hr. A splenectomy before the PCl elicitation significantly reduced the ALT elevation. Both SPC and NPC from liver injury mice induced a remarkable release of ALT from HC in vitro, in parallel with their LFA-1 expression. The pre-treatment of NPC or SPC with anti-LFA-1 mAb, irrespective of the presence of complement, completely blocked the ALT release. Also, when HC was prebound with anti-ICAM-1 mAb, neither NPC nor SPC showed a cytotoxicity against the HC. Furthermore, the treatment of NPC with either anti-Thy1.2 or anti-CD4 mAb in the presence but not absence of complement, showed a complete abolishment of ALT release. Anti-CD8 mAb plus complement also tended to inhibit ALT release. The twofold increase in CD4+ LFA-1+ and mild increase in CD8+ LFA-1+ populations were also confirmed in NPC at 12 hr. These results suggest that PCl elicitation in liver may trigger an increased expression of LFA-1 on SPC and NPC and ICAM-1 on HC. LFA-1/ICAM-1 interaction between liver-infiltrating NPC, mainly including CD4+ and CD8+ T cells, and HC may be an essential step for the hepatocyte damage in PCl-DTH liver injury.

Alanine Transaminase↗

PubMed1998

Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line.

Fowl adenoviruses, many of which appear to be non-pathogenic, are ubiquitous in birds. In addition, the genome of these viruses is large, making them ideal candidates for construction as vectors for foreign genes. Current methods to cultivate fowl adenoviruses use primary cell cultures derived from embryonated chicken eggs. In order to provide a more suitable culture method, the growth of fowl adenovirus type 8 (FAdV-8) was investigated in CH-SAH, a continuous hepatoma cell line. A one step growth curve demonstrated release of extracellular virus beginning by 18 h p.i. and with a final yield about 100 fold higher than that in chicken embryo liver cells. Viral DNA synthesis was first detected 8 h prior to this. The CH-SAH cell line supported the production of progeny viruses similar to the wild-type virus after being transfected with purified FAdV-8 DNA. This study demonstrated that the continuous hepatoma cell line is an appropriate in vitro host for FAdV-8.

Animals↗

PubMed1998

Safety evaluation and fluorine concentration of Pu'er brick tea and Bianxiao brick tea.

Pu'er brick tea and Bianxiao brick tea are both compressed types of tea. Fluorine analysis was carried out on samples of Pu'er brick tea produced at different times in Yunnan Province and on samples of Bianxiao brick tea made in Hunan and Sichuan Province for supply especially to minority ethnic groups in border areas of China. The levels of water-soluble and water-insoluble fluorine were measured in the tea samples using an ion-specific electrode potentiometer. The concentration of water-soluble fluorine was much greater in Bianxiao brick tea than in Pu'er brick tea (mean levels 441 and 77 mg/kg, respectively). According to these figures, the fluorine intake associated with consuming an infusion of 30 g Pu'er brick tea/person/day is safe because it does not exceed the maximum recommended daily allowance (RDA) of up to 4.0 mg for adults. In contrast, the almost six times higher intake of fluorine from Bianxiao brick tea greatly exceeds the 4 mg RDA and is unsafe. The difference in the fluorine levels of the two types of brick tea can be attributed to differences in the materials used to make them: Pu'er brick tea is made from tender leaves whereas Bianxiao brick tea is made from old tough leaves in which fluorine has accumulated. We conclude that consumption of Pu'er brick tea is unlikely to induce fluorosis, which has been associated with consumption of Bianxiao brick tea.

Fluorine↗

PubMed1998

Ethylene biosynthesis: processing of a substrate analog supports a radical mechanism for the ethylene-forming enzyme.

BACKGROUND: The chemical mechanism of the final step of ethylene biosynthesis (the conversion of 1-aminocyclopropanecarboxylic acid, ACC, to ethylene by ACC oxidase, the ethylene-forming enzyme, EFE) is poorly understood. Two possibilities have been suggested: a radical mechanism and an N-hydroxylation mechanism. We investigated reaction pathways available to radical intermediates in this reaction using an ACC analog, 1-aminocyclobutanecarboxylic acid (ACBC) as a substrate. RESULTS: ACBC was converted to dehydroproline (delta 1-pyrroline-2-carboxylic acid) by the EFE via a ring expansion process. The possibility that an N-hydroxy-aminoacid (produced during two-electron oxidation) acts as an intermediate in this process was eliminated by control experiments. Chemical model reactions involving two-electron oxidants, such as a positive halogen (X+), which presumably generate N-halo derivatives, produce only decarboxylation products. Radical-based oxidants, in contrast, generate dehydroproline. Model reactions involving sequential single-electron transfer mechanisms also produce dehydroproline; thus our results support the proposal that the EFE-catalyzed step of ethylene biosynthesis proceeds using a radical-based mechanism. CONCLUSIONS: Our results provide support for a radical mechanism in the final step of ethylene biosynthesis and refute an alternative N-hydroxylation mechanism. This work extends the idea that the intrinsic chemical reactivity of a high energy iron-oxo intermediate can account for the observed products in ethylene biosynthesis.

Amino Acids↗

PubMed1998

Autologous CD34-selected blood progenitor cell transplants for patients with advanced multiple myeloma.

Fifty-five patients with advanced multiple myeloma received purified CD34-selected peripheral blood progenitor cell transplants following myeloablative chemotherapy. A median of 4.1 x 10(6) CD34 cells/kg (range 1.2-30.7) were infused after busulfan (14 mg/kg) and cyclophosphamide (120 mg/kg); granulocyte-macrophage colony-stimulating factor was used until hematopoietic recovery. Median time to neutrophils >0.5 x 10(9)/l and platelets >20 x 10(9)/l were 12 days (range 10-16 and 8-184 days, respectively). Median follow-up of survivors from the time of transplantation is 33 months (range 7 to 44 months). Thirty-one patients are alive, 19 progression-free. Median progression-free survival is 14 months. Actuarial 3-year progression-free and overall survival are 29+/-14% and 47+/-17%. CD34-selection of peripheral blood progenitor cells provides effective hematopoietic support with significant progression-free and overall survival.

Adult↗

PubMed1998

Method for reduction of inhibition in a Mycobacterium tuberculosis-specific ligase chain reaction DNA amplification assay.

The present study describes the identification of inhibitors of a Mycobacterium tuberculosis-specific gap ligase chain reaction (LCR) DNA amplification assay as well as a method for their removal. A major contributor to inhibition was deduced to be a calcium phosphate precipitate, CaHPO4. The precipitate forms during N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) decontamination, digestion, and concentration of respiratory specimens. The solubility product of CaHPO4 precipitate at pH 7.8, the pH at which gap LCR is optimized, indicates that the precipitate releases an amount of phosphate ions sufficient to inhibit amplification. A method for removal of the precipitate was identified. The precipitate is dissociated by exposing it to a mildly acidic (pH 4.1) buffer during the first of two centrifugation steps; the inhibitory phosphate ions are removed by the centrifugation steps. When 100 NALC-NaOH respiratory sediments were tested by gap LCR, none of the sediments were inhibitory when the acidic buffer was used while 24 samples were inhibitory when TE buffer, pH 7.8, was used. In another study, when the acidic buffer wash was applied to 1,440 NALC-NaOH respiratory sediments, only 10 sediments were found to be inhibitory. None of the inhibited sediments were culture positive for M. tuberculosis. This work demonstrates that when inhibition mechanisms are identified, relatively simple protocols can be used to obtain low inhibition rates and to allow the use of larger volume equivalents in amplification reactions.

Acetylcysteine↗