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Biomedical subjects

D Ma

Publications and source records attributed to D Ma.

At least 217 records · Page 12Linked to original sources
PubMed1995

Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli.

Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules. Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these beta-lactams across the outer membrane was not increased. This finding is inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi-drug efflux pump. The natural environment of an enteric bacterium such as E. coli is enriched in bile salts and fatty acids. An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate. In addition, acrAB expression was elevated by growth in 5 mM decanoate. These results suggest that one major physiological function of AcrAB is to protect E. coli against these and other hydrophobic inhibitors. Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 M NaCl, and stationary phase in Luria-Bertani medium. Finally, acrAB expression was shown to be increased in mar (multiple-antibiotic-resistant) mutants.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Neuronal expression of human immunodeficiency virus type 1 env proteins in transgenic mice: distribution in the central nervous system and pathological alterations.

It is now well documented that human immunodeficiency virus type 1 (HIV-1) induces encephalopathy in patients with AIDS. In vitro studies have implicated the envelope protein (gp120) as a factor which causes neuronal death. To better evaluate the role and elucidate the mechanisms of gp120 neurotoxicity, we have developed transgenic mice carrying a segment of the HIV-1 genome that expresses the viral gp160 protein under the control of the human neurofilament light gene promoter. In two separate lines of transgenic mice, the Env protein was found to be expressed in several nuclei of the brain stem and in the anterior horns of the spinal cord. The two lines showed identical patterns of Env expression. Neuropathological evaluation revealed numerous abnormal dendritic swellings in the immunostained motor neuron structures. Large and numerous neuritic swellings were also prominent in the nucleus gracilis and in the gracilis and cuneate fascicles. In addition, reactive astrocytosis was observed in several immunoreactive areas of the central nervous system. These transgenic mice offer a unique model to further investigate the role of HIV-1 Env protein in neuronal toxicity and to help elucidate the mechanisms that are involved.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Tissue-type plasminogen activator-induced invasion and metastasis of murine melanomas.

The role of tissue-type plasminogen activator (tPA) in the 'spontaneous' as well as 'experimental' metastasis of ocular melanomas in mice was evaluated by transfecting the D5.1G4 murine melanoma cell line that possesses low metastatic activity and low tPA activity with a full length cDNA encoding human tPA. For comparison, a highly metastatic melanoma cell line (Queen's) that constitutively expresses high tPA production, was transfected with a cDNA coding for human plasminogen activator inhibitor type 1 (PAI-1). Unlike non-transfected controls, transfected D5.1G4 melanoma cells expressed high levels of tPA and produced extensive pulmonary metastases following intravenous injection. By contrast, PAI-1 transfected Queen's melanoma cells expressed low tPA activity and displayed significantly reduced metastatic potential compared with nontransfected controls. Moreover, PAI-1 transfected Queen's melanoma cells did not metastasize from the eye while nontransfected parental cells produced extensive spontaneous metastases. Expression of tPA activity in transfected and nontransfected cell lines was completely blocked by an anti-tPA antibody. This antibody significantly inhibited the organ localization and frequency of lung metastases of both Queen's and tPA-transfected D5.1G4 melanomas. This study demonstrates that tPA is involved in the metastasis of murine intraocular melanomas.

Animals↗
PubMed1995

Transforming growth factor-beta down-regulates major histocompatibility complex class I antigen expression and increases the susceptibility of uveal melanoma cells to natural killer cell-mediated cytolysis.

Intraocular melanomas, especially those of the anterior segment, reside within an immunologically privileged milieu. Aqueous humour contains a variety of immunomodulatory factors that are believed to contribute to ocular immune privilege. Among these is transforming growth factor-beta (TGF-beta), which has been shown to down-regulate major histocompatibility complex (MHC) class I antigens on normal cells. Since the susceptibility of tumour cells to natural killer (NK) cell-mediated lysis is inversely correlated with the expression of MHC class I antigens, tumour cells exposed to TGF-beta might be expected to experience enhanced susceptibility to NK-mediated killing. This was examined by incubating two human uveal melanoma cell lines in the presence of TGF-beta and evaluating the expression of MHC class I antigen and susceptibility to NK cell-mediated lysis. OCM1 and OCM8 melanoma cells constitutively express high levels of class I antigen (85-90% positive) and low susceptibility to NK-mediated lysis in vitro (3-8%). Incubation with TGF-beta produced a significant reduction in class I antigen expression (52-62%) and a proportional increased susceptibility to NK cell-mediated cytolysis (17%). Analogous effects were found using a human uveal melanoma cell line (OCM3) that constitutively expresses low amounts of class I (< 5% positive) and high NK susceptibility (35% lysis). Stimulation of class I antigen expression by incubation with interferon-gamma resulted in a sharp increase in class I expression (80% positive) and a comparable diminution in susceptibility to NK cell-mediated lysis (< 10%). The results indicate that TGF-beta, at concentrations found in the aqueous humour, can significantly alter MHC class I antigen expression and the susceptibility of ocular melanoma cells to NK cell-mediated cytolysis.

Animals↗
PubMed1995

Effect of alteration of the heterocyclic nucleus of ILV on its isoform selectivity for PKC. Palladium catalyzed route to benzofuran analogues of ILV.

A palladium catalyzed route for the preparation of several benzofuran analogues of the PKC activator indolactam V (ILV) is described together with the ability of these compounds to activate the isoforms of PKC. The benzofuran analogues of ILV are shown to activate PKC with a slightly different pattern of isotype selectivity than ILV or 7-n-octyl-ILV. Moreover, in an examination of the effect of stereochemistry at the C-14 center of the teleocidins on PKC binding activity, a clear preference for R-stereochemistry at the C-14 center was found, thus providing additional verification of previously published structural correlations between the families of PKC activators.

Benzofurans↗
PubMed1995

Efficacy of tumor-infiltrating lymphocytes in the treatment of hepatic metastases arising from transgenic intraocular tumors in mice.

PURPOSE: The purpose of this study was to determined if tumor-infiltrating lymphocytes (TIL) isolated from transgenic intraocular tumors were capable of preventing the development of spontaneous hepatic metastases. METHODS: Tumor-infiltrating lymphocytes were isolated from intraocular tumors in immunocompetent mice and were examined for cytolytic activity in vitro. The antigenic phenotypes of the TIL were determined by in situ immunohistology. Cultured TIL were transferred adoptively to immunoincompetent, intraocular, tumor-bearing recipients to determine the efficacy of TIL in preventing spontaneous liver metastases. RESULTS: Cultured TIL displayed remarkable cytolytic activity in vitro and antimetastatic properties after adoptive transfer into immunoincompetent, athymic nude mice and anti-CD4/CD8-treated euthymic FVB/N mice. Hepatic metastases developed in only 2 of 10 athymic nude mice that subsequently received TIL, whereas metastases developed in all 10 control mice. Similar results were found in anti-CD4/CD8-treated FVB/N mice. Hepatic metastases did not develop in any of the 10 mice treated with TIL, whereas extensive metastatic foci developed in all 10 untreated FVB/N control mice. The efficacy of TIL therapy was manifested also by significant prolongation of host survival times. CONCLUSIONS: Tumor-infiltrating lymphocytes isolated from intraocular tumors can be expanded in vitro and can exert antimetastatic effects in vivo.

Animals↗
PubMed1995

Successful immunization against Acanthamoeba keratitis in a pig model.

The feasibility of inducing protective immunity to Acanthamoeba keratitis was tested in a pig model. Experiments were designed to determine if ocular infection with Acanthamoeba trophozoites would elicit protection against reinfection. Additional experiments examined whether injection of parasite antigens either intramuscularly, subconjunctivally, or by both routes would induce immunity. Therefore, four groups of animals were examined: (a) pigs that had resolved a primary corneal infection with Acanthamoeba; (b) pigs immunized intramuscularly; (c) pigs immunized subconjunctivally; and (d) pigs immunized intramuscularly and subconjunctivally. Animals were subsequently challenged with parasite-laden soft contact lenses and observed clinically for the appearance of Acanthamoeba keratitis. Acanthamoeba-specific serum antibody titers and blastogenic responses of peripheral blood lymphocytes were determined weekly. The results indicated that intramuscular injection of Acanthamoeba antigens failed to protect against ocular infection even though hosts developed high titers of IgG antibodies and displayed lymphocyte blastogenic responses to parasite antigens. Ocular infection alone failed to stimulate immunity in any of the animals. By contrast, 50% of the hosts immunized subconjunctivally were protected against corneal disease, and 100% of the animals immunized by a combination of intramuscular and subconjunctival administration of parasite antigens were completely protected against two separate ocular challenges with infectious parasites. Protection did not correlate with either IgG antibody titers or blastogenic potentials of peripheral blood lymphocytes. Interestingly, ocular infection alone failed to stimulate immunity to subsequent ocular challenge with infectious parasites. Thus, administration of parasite antigen via the subconjunctival route can protect against Acanthamoeba keratitis.

Acanthamoeba↗
PubMed1995

[Hemofiltration ameliorating reperfusion injury after complete cerebral ischemia: an experimental study].

The effects of hemofiltration on cerebral resuscitation were studied by using resuscitation model of potassemia-induced cardiac arrest and cardiopulmonary bypass. Sixteen rabbits were divided into control group (CG) and hemofiltration group (HFG). Blood lactic acid, lipid peroxidant (LPO), serum iron, middle molecular substance (MMS) were determined. Neurologic deficit score (NDS) system was used to evaluate neurologic function. The level of blood lactic acid was increased in both CG and HFG. It was higher in CG than in HFG (P > 0.05). Serum iron was increased in CG, however, it was decreased in HFG. The difference between the two groups was significant (P < 0.01). LPO was increased significantly in CG and there was no obvious changes in HFG. MMS was increased gradually during reperfusion in CG, whereas it was decreased in HFG. There was significant difference in the corresponding times of the two groups. NDS of CG was higher than that of HFG (32 +/- 12 vs 18 +/- 10) (P < 0.01). These data demonstrate that hemofiltration is able to eliminate ischemic and reperfusional toxic products quickly. Additionally, free iron, agonist factor of catalyzing free radical reaction, does not increase because free radicals and lactic acid are eliminated quickly, inhibiting free radical tissue injury and improving cerebral resuscitation.

Animals↗
PubMed1995

Relationship between natural killer cell susceptibility and metastasis of human uveal melanoma cells in a murine model.

PURPOSE: The purpose of this study was to determine the susceptibility of human uveal melanoma cells to in vitro and in vivo natural killer (NK) cell-mediated cytolysis and to determine if NK cells influence metastasis from the eye. METHODS: Four human uveal melanoma cell lines and one melanoma cell line derived from a metastatic lesion from a patient with uveal melanoma were tested for in vitro and in vivo NK cell-mediated lysis in a mouse model. Major histocompatibility complex (MHC) class I antigen expression was evaluated by flow cytometry. The role of NK cells in controlling the metastasis of uveal melanoma cells from the eye to the liver was examined in nude mice. RESULTS: Sensitivity to in vitro and in vivo lysis by human and murine NK cells was correlated with reduced expression of MHC class I antigens. Uveal melanoma lines expressing normal MHC class I antigen expression were insensitive to NK cell-mediated lysis, both in vitro and in vivo. Metastasis of uveal melanoma cells was inhibited by NK cell activity because disruption of in vivo NK function produced a sharp increase in the spontaneous metastasis of intraocular melanomas in nude mice. CONCLUSIONS: There is considerable variation in the susceptibility of human uveal melanomas to NK cell-mediated cytolysis. Susceptibility is closely correlated with reduced expression of MHC class I antigen expression. Disruption of NK cell function significantly increases the development of hepatic metastases from human uveal melanoma cells.

Animals↗
PubMed1995

[Pathological studies on the anti-invasive character of IL-6 gene transfected leukemia cells].

FBL-3 Leukemia cells transfected with IL-6 gene were expanded in vitro and inoculated into C57BL/6 mice subcutaneously. Tumor growth was observed and histologic analyses of the tumors in situ and the liver, spleen and bone marrow were performed at 14, 21, 28 and 35 days after inoculation. The mice inoculated with wild-type FBL-3 leukemia cells were used as the control. We found that the tumor invasiveness in the mice inoculated with FBL-3-IL-6+ occurred later than in the control group. The survival time of experimental mice was longer than in the control mice. The results demonstrated that inoculation of IL-6 high-secreting FBL-3 inhibited invasiveness of the leukemia cells, suggesting that the IL-6 gene transfected FBL-3 cells can be used as a vaccine to treat leukemia. The mechanism of the anti-invasiveness of IL-6 gene transfected leukemia cells needs further study.

Animals↗
PubMed1994

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription.

Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells.

Amino Acid Sequence↗
PubMed1994

Efficient anchoring of RNA polymerase in Escherichia coli during coupled transcription-translation of genes encoding integral inner membrane polypeptides.

While it has been known that supercoiling of the DNA template can be induced by transcription, the mechanism and the efficiency of this process in vivo is not fully understood. We report here that transcription of genes encoding 16 S rRNA, a stable RNA species, or cytoplasmic polypeptides leads to very little or no detectable DNA supercoiling even under the optimum conditions in Escherichia coli. This indicates that hydrodynamic drag on the transcription complex (including RNA polymerase, nascent RNA, ribosomes, and nascent polypeptides) is not sufficient to anchor RNA polymerase during coupled transcription-translation. On the other hand, transcription of membrane-associated genes encoding integral inner membrane or exported periplasmic polypeptides leads to apparent DNA supercoiling. Transcription of genes encoding integral inner membrane polypeptides leads to significantly greater anchoring of RNA polymerase than does transcription of genes encoding periplasmic polypeptides. This may reflect differences in the coupling of transcription-translation with membrane association during expression of these two classes of polypeptides. Evidence is further presented to suggest that the anchoring of RNA polymerase is probably achieved through the interaction of nascent polypeptides with the cytoplasmic surface of the inner membrane during coupled transcription-translation. Moreover, transcriptions of a membrane-associated gene can, under certain circumstances, induce topological anchoring of an RNA polymerase transcribing a neighboring gene that ordinarily is not membrane-associated. Finally, the potential biological consequences of our findings are discussed.

Bacterial Proteins↗
PubMed1994

Capacity of simian virus 40 T antigen to induce self-tolerance but not immunological privilege in the anterior chamber of the eye.

Transgenic mice bearing the simian virus 40 (SV40) large T oncogene developed progressively growing intraocular tumors and displayed characteristics of immunological tolerance to SV40 T antigen. Transgenic mice failed to mount CTL responses to SV40 T antigen-bearing tumor cell lines derived from the transgenic intraocular tumors. Spleen cells from transgenic hosts were able to prevent the in vivo and in vitro generation of CTL responses by lymphocytes from normal syngeneic FVB/N mice. Adoptive transfer of spleen cells from tolerant transgenic donors temporarily inhibited the immunological rejection of SV40 T antigen-positive tumor cells transplanted to normal syngeneic FVB/N recipients. Thus, introduction of SV40 transforming sequences into the mouse germline induced tolerance to SV40 T antigen. However, in normal FVB/N mice, SV40 T antigen-bearing tumor cells failed to experience immune privilege in the anterior chamber and did not elicit systemic down-regulation of delayed-type hypersensitivity responses that characteristically occur when antigens are introduced into the anterior chamber. The results indicate that within the anterior chamber of the eye, SV40 T antigen-bearing cells are perceived by the host's immune system much differently than are other categories of antigen. Thus, SV40 T antigen effectively induces self-immunological tolerance when its gene is introduced into the host's germline but fails to experience immunological privilege in the anterior chamber of the eye in normal hosts.

Animals↗
PubMed1994

Isolation and characterization of gap junctions in the osteoblastic MC3T3-E1 cell line.

Gap junctions are channels connecting cells that function in cell-to-cell communication. Gap junctions are abundant in osteoblastic cells. Membranes enriched for gap junction plaques were obtained by differential centrifugation, followed by treatment of the membranes with potassium iodide and sarkosyl before sucrose density gradient centrifugation. Electron microscopy showed that the preparation was enriched for electron-dense membranes consistent with gap junctions. Coomassie Blue staining of SDS-PAGE preparations revealed a prominent band at approximately 41 kD. Western analysis with a site-directed antibody, CT-360 (D. Laird, California Institute of Technology, Pasadena, CA), to the C-terminal portion of the rat heart connexin 43 molecule was positive in the MC3T3-E1 cell line, a phenotypic osteoblastic cell line derived from normal neonatal mouse calvariae. Western analysis using a monoclonal antibody, R5.21C, to rat liver connexin 32 was negative. Additionally, a prominent band at 59 kD was detected by CT-360 in both gap junction-enriched preparations and cell lysates. Treatment of diluted samples of gap junction-enriched preparations with sulfhydryl reducing agents in combination with detergents resulted in the enhancement and diminution of the 41 and 59 kD bands, respectively. Immunoprecipitation following [35S]methionine/[35S]cysteine labeling revealed a significant band detected at 122 kD in addition to the 41 kD band. To demonstrate functional gap junctions, transfer of lucifer yellow dye to surrounding cells was monitored after microinjection of a target cell. Between passages 10 and 25 in culture, functional cell coupling was found in approximately 70% of injected cells. Coupling was detected within 1-2 minutes after injection. Simultaneous microinjection of the CT-360 antibody with lucifer yellow resulted in the decoupling of cells. In conclusion, (1) MC3T3-E1 cells possess a 41 kD protein that is recognized by connexin 43 antibody to rat heart gap junction; (2) multimers of the MC3T3-E1 gap junctions occur in the preparation; and (3) functional coupling demonstrated by dye transfer may be regulated by region(s) in the C terminus of the connexin molecule.

3T3 Cells↗
PubMed1994

Establishing a resuscitation model in rabbits with closed-thoracic cardiopulmonary by-pass.

This study aims to establish an animal model of resuscitation in rabbits by using closed-thoracic cardiopulmonary by-pass (CTCPB). The rabbits were randomly divided into four groups according to cardiac arrest times which were 8, 10, 12, and 15 min. Neurologic outcome and blood lactate were determined within 150 min after resuscitation. It was indicated that the rabbits' neurologic functions were progressively injured with prolonged cardiac arrest time. The threshold of circulatory arrest time that induced a vegetative state in the rabbits was between 10 and 12 min. There were no significant differences in cardiac resuscitability among the four groups, as was so for plasma lactate, although it increased significantly from the control levels. The establishment of a small-animal model of resuscitation by using CTCPB, and the problems in dealing with it are also described and discussed in detail in this paper. Our experience indicated that this is a simple, convenient, and economical animal model for the study of resuscitation.

Animals↗
PubMed1994

Efflux pumps and drug resistance in gram-negative bacteria.

The outer membrane of Gram-negative bacteria can only slow down the influx of lipophilic inhibitors, and so these bacteria need active efflux pumps of broad specificity to survive. Pumps such as the Escherichia coli Acr system and its homologs make Gram-negative bacteria resistant to dyes, detergents and antibiotics.

Anti-Bacterial Agents↗
PubMed1994

Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to beta-lactam resistance.

Wild-type strains of Pseudomonas aeruginosa are more resistant to various beta-lactam antibiotics as well as other agents than most enteric bacteria. Although resistance to compounds of earlier generations is explained by the synergism between the outer membrane barrier and the inducible beta-lactamase, it was puzzling to see significant levels of resistance to compounds that do not act as inducers or are not hydrolyzed rapidly by the chromosomally encoded enzyme. This intrinsic-resistance phenotype becomes enhanced in those strains with the so-called intrinsic carbenicillin resistance. In the accompanying paper (X.-Z. Li, D. M. Livermore, and H. Nikaido, Antimicrob. Agents Chemother. 38:1732-1741, 1994), we showed that active efflux played a role in the resistance, to various non-beta-lactam agents, of P. aeruginosa strains in general and that the efflux was enhanced in intrinsically carbenicillin-resistant strains. We show in this paper that, in comparison with the drug-hypersusceptible mutant K799/61, less benzylpenicillin was accumulated in wild-type strains of P. aeruginosa and that the accumulation levels were even lower in intrinsically carbenicillin-resistant strains. Deenergization by the addition of a proton conductor increased the accumulation level to that expected for equilibration across the cytoplasmic membrane. In intrinsically carbenicillin-resistant isolates, there was no evidence that either nonspecific or specific permeation rates of beta-lactams across the outer membrane were lowered in comparison with those of the more susceptible isolates. Furthermore, these carbenicillin-resistant isolates were previously shown to have no alteration in the level or the inducibility of beta-lactamase and in the affinity of penicillin-binding proteins. These data together suggest the involvement of an active efflux mechanism also in the resistance to beta-lactams. Hydrophilic beta-lactams with more than one charged group did not cross the cytoplasmic membrane readily. Yet one such compound, ceftriaxone, appeared to be extruded from the cells of more-resistant strains, although with this compound effects of proton conductors could not be shown. We postulate that wild-type strains of P. aeruginosa pump out such hydrophilic beta-lactams either from the periplasm or from the outer leaflet of the lipid bilayer of the cytoplasmic membrane, in a manner analogous to that hypothesized for multidrug resistance protein of human cancer cells (M.M. Gottesman and I. Pastan, Annu. Rev. Biochem. 62:385-427, 1993).

Anti-Bacterial Agents↗