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Biomedical subjects

M C Walker

Publications and source records attributed to M C Walker.

At least 91 records · Page 5Linked to original sources
PubMed1991

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding.

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.

Electron Transport↗
PubMed1991

Laser flash photolysis studies of the kinetics of reduction of ferredoxins and ferredoxin-NADP+ reductases from Anabaena PCC 7119 and spinach: electrostatic effects on intracomplex electron transfer.

The influence of electrostatic forces on the formation of, and electron transfer within, transient complexes between redox proteins was examined by comparing ionic strength effects on the kinetics of the electron transfer reaction between reduced ferredoxins (Fd) and oxidized ferredoxin-NADP+ reductases (FNR) from Anabaena and from spinach, using laser flash photolysis techniques. With the Anabaena proteins, direct reduction by laser-generated flavin semiquinone of the FNR component was inhibited by complex formation at low ionic strength, whereas Fd reduction was not. The opposite results were obtained with the spinach system. These observations clearly indicate structural differences between the cyanobacterial and higher plant complexes. For the complex formed by the Anabaena proteins, the results indicate that electrostatic forces are not a major contributor to complex stability. However, the rate constant for intracomplex electron transfer had a biphasic dependence on ionic strength, suggesting that structural rearrangements within the transient complex facilitate electron transfer. In contrast to the Anabaena complex, electrostatic forces are important for the stabilization of the spinach Fd:FNR complex, and changes in ionic strength had little effect on the limiting rate constant for intracomplex electron transfer. This suggests that in this case the geometry of the initial collisional complex is optimal for reaction. These results provide a clear illustration of the differing roles that electrostatic interactions may play in controlling electron transfer between two redox proteins.

Cyanobacteria↗
PubMed1990

Inherent sensitivity and induced resistance to chemotherapeutic drugs and irradiation in human cancer cell lines: relationship to mutation frequencies.

Metastatic nonseminomatous testicular germ cell tumors are curable using combination chemotherapy in approximately 80% of patients. In contrast, most other patients with other types of cancer either present with or acquire drug-resistant disease following chemotherapy. Cell lines derived from testis tumors retain hypersensitivity to both drugs and radiation in vitro, thus providing a model system with which to investigate the genetic basis of hypersensitivity to these agents. This study compared the spontaneous and both ethyl methanesulfonate- and cisplatin-induced frequencies of mutation of 6-thioguanine resistance in 3 human bladder and 3 testis tumor cell lines and a bladder and a testis cell line with cisplatin resistance induced in vitro. The two tumor types showed similar frequencies of both spontaneous and induced mutation frequencies at this locus. Therefore, we failed to provide evidence for the hypothesis that the curability of testis tumors is associated with a low frequency of mutation to drug resistance.

Antineoplastic Agents↗
PubMed1990

Comparison of the kinetics of reduction and intramolecular electron transfer in electrostatic and covalent complexes of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119.

The kinetics of reduction and intracomplex electron transfer in electrostatically stabilized and covalently crosslinked complexes between ferredoxin-NADP+ reductase (FNR) and flavodoxin (Fld) from the cyanobacterium Anabaena PCC 7119 were compared using laser flash photolysis. The second-order rate constant for reduction by 5-deazariboflavin semiquinone (dRfH) of FNR within the electrostatically stabilized complex at 10 mM ionic strength (4.0 X 10(8) M-1 s-1) was identical to that for free FNR. This suggests that the FAD cofactor of FNR is not sterically hindered upon complex formation. A lower limit of approximately 7000 s-1 was estimated for the first-order rate constant for intracomplex electron transfer from FNRred to Fldox under these conditions. In contrast, for the covalently crosslinked complex, a smaller second-order rate constant (2.1 X 10(8) M-1 s-1) was obtained for the reduction of FNR by dRfH within the complex, suggesting that some steric hindrance of the FAD cofactor of FNR occurs due to crosslinking. A limiting rate constant of 1000 s-1 for the intracomplex electron transfer reaction was obtained for the covalent complex, which was unaffected by changes in ionic strength. The substantially diminished limiting rate constant, relative to that of the electrostatic complex, may reflect either a suboptimal orientation of the redox cofactors within the covalent complex or a required structural reorganization preceding electron transfer which is not allowed once the proteins have been covalently linked. Thus, although the covalent complex is biochemically competent, it is not a quantitatively precise model for the catalytically relevant intermediate along the reaction pathway.

Cyanobacteria↗
PubMed1990

Development and characterization of cisplatin-resistant human testicular and bladder tumour cell lines.

Cisplatin-resistant cells were derived in vitro from a human bladder carcinoma cell line (RT112) and a testicular tumour cell line (SuSa) by continuous exposure to increasing concentrations of cisplatin for 14 and 11 months, respectively. Both resistant cell lines had a four-fold level of resistance relative to their parental cell lines, comparing the cisplatin concentration to inhibit colony forming ability by 70%. These levels of resistance were retained in the absence of cisplatin for at least 3 months. In each case, four-fold fewer micronuclei were produced in the resistant lines by the same cisplatin concentrations. Cross-resistance to carboplatin and methotrexate was observed in both resistant cell lines, but neither line was resistant to doxorubicin. Isozyme and DNA analysis with hypervariable probes confirmed the origin of each resistant cell line from its parental line. Population doubling times and intermitotic times were similar in each of the pairs of cell lines. Karyotyping showed that the resistant cell lines had gained and lost marker chromosomes, but there were no changes common to both resistant cell lines.

Carcinoma, Transitional Cell↗
PubMed1990

5-azacytidine and 5-azadeoxycytidine inhibit human immunodeficiency virus type 1 replication in vitro.

Chemotherapeutic agents which affect the integration, stability, or inducibility of the human immunodeficiency virus (HIV) provirus would have considerable value in treating acquired immunodeficiency syndrome. Two nucleoside analogs of cytosine, 5-azacytidine and 5-azadeoxycytidine, which seem to have such value because of their capabilities to affect both the stability and the methylation patterns of the nucleic acids into which they are incorporated, were tested for their ability to inhibit the replication of HIV type 1 (HIV-1) in human CEM T cells in vitro. 5-Azadeoxycytidine (1 microM) completely inhibited HIV replication in CEM cells, by the criteria of reduced viral antigen expression and decreased supernatant reverse transcriptase activity, with little toxicity for the treated cells. 5-azacytidine (1 microM) also inhibited HIV replication, but less effectively. When added 2 or more h after CEM cells were infected with HIV-1, both 5-azacytosine derivatives were less effective than they were when added at the time of infection. Even 2 h of exposure to 5-azadeoxycytidine was sufficient for inhibition of HIV replication. Although long exposure to either analog at concentrations of 1 microM would result in pronounced cellular cytotoxicity, the the fact that short exposures to the same dose of drug inhibit HIV replication but are not toxic for the cells implies that cellular toxicity itself is not an important mechanism of the antiviral action of the analogs.

Antineoplastic Agents↗
PubMed1990

Anti-HIV antibodies and other serological and immunological parameters among normal Haitians in Montreal.

A matched-pair, cross-sectional study of lymphocyte and serological parameters associated with acquired immune deficiency syndrome (AIDS) in 189 randomly chosen, ostensibly healthy adult Haitian immigrants residing in Montreal matched for sex, age (within 5 years), and neighborhood of residence to 189 non-Haitian (Caucasian) controls was done in 1983-1984. Three years later (1986-1987), 41 of the Haitian study subjects and 83 of the non-Haitian controls participated in a follow-up study centered on lymphocyte parameters. A significantly greater number of Haitians than controls had produced antibodies to Toxoplasma gondii. In addition, a greater percentage of the Haitians than the controls were also producing antibodies to two other opportunistic pathogens frequently encountered in AIDS, cytomegalovirus and hepatitis B virus, implying that the Haitians in general had had greater exposure to a variety of infectious agents than had the controls. A few study participants were producing antibodies against two viruses that are related to the human immunodeficiency virus-type 1 (HIV-1), the human T-cell lymphotropic viruses I and II (HTLV-I and -II). Two Haitians and one control were producing antibodies against HTLV-I. One study subject and four controls were HTLV-II seropositive. The most interesting and surprising finding was that four (2.1%) of the Haitian study subjects but none of the controls were seropositive for HIV-1. These individuals, two of whom were women and two men, were asymptomatic. Although their individual lymphocyte parameter values fell in the normal range, as a group they had statistically significantly lower average values for their lymphocyte parameters than did the HIV-seronegative Haitian study objects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Effects of antibody to rotavirus on protection of adults challenged with a human rotavirus.

Effects of preinoculation rotavirus antibody titers on the probability of infection and illness were evaluated in adults challenged orally with different doses of a virulent human rotavirus (CJN strain). Preinoculation titers considered were serum neutralizing antibody, serum rotavirus IgA, serum rotavirus IgG, jejunal neutralizing antibody, jejunal rotavirus IgA, and stool rotavirus IgA. Doses of virus of either 9 x 10(1) or 9 x 10(3) focus-forming units were administered to 19 subjects each. Twenty-six were infected; 15 experienced illness. The probability of either outcome was unrelated to dose. Stool rotavirus IgA titers could not be correlated to either infection or illness, but the mean titers of the other five antibodies were significantly or nearly significantly lower in subjects infected or ill, when compared with those negative for either outcome. When analyzed by stepwise logistic regression, only serum rotavirus IgG remained significantly (P = .005) related to the probability of infection, and only jejunal neutralizing antibody remained significantly (P = .01) related to the probability of illness.

Adult↗
PubMed1989

Use of a holding technique to control the violent behavior of seriously disturbed adolescents.

Therapeutic holding is a treatment technique in which a violent patient is physically contained by people rather than by mechanical or chemical restraints or seclusion. Episodes of therapeutic holding that took place on an inpatient adolescent psychiatric unit over an 18-month period were examined retrospectively through chart review and review of written audits of each episode. During that period, 22.8 percent of the patient population experienced at least one episode of therapeutic holding. Patients requiring this intervention were more likely to be younger and, when day at risk were controlled, to be male and to have had longer stays on the unit. The technique enabled patients to regain behavioral control after a mean of 21.2 minutes, a much shorter period of containment than the four to 16 hours reported in studies of seclusion.

Adolescent↗
PubMed1988

Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines.

The formation and removal of four platinum-DNA adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (RT112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 microM (5 micrograms/ml) cisplatin. RT112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinated DNA may account for their hypersensitivity to cisplatin.

Cell Line↗
PubMed1988

Intravesical chemotherapy: combination with dimethyl sulfoxide does not enhance cytotoxicity in vitro.

There is evidence that dimethyl sulfoxide (DMSO) can increase the anticancer activity of chemotherapeutic drugs. As DMSO is instilled into the bladder for interstitial cystitis, it could be readily adopted in clinical practice if it was found to enhance the effectiveness of the drugs used for intravesical chemotherapy. The purpose of this study was to investigate, using a human bladder cancer cell line, the hypothesis that DMSO enhances the activity of these agents. However, the addition of 4% DMSO to the four drugs most frequently used for intravesical chemotherapy (adriamycin, epodyl, mitomycin-c, thiotepa) did not increase tumour cell kill in vitro.

Administration, Intravesical↗
PubMed1987

Differential expression of collateral sensitivity or resistance to cisplatin in human bladder carcinoma cell lines pre-exposed in vitro to either X-irradiation or cisplatin.

Two sublines were derived from a human bladder carcinoma continuous cell line (RT112-P), one by exposure to fractionated X-irradiation (RT112-DXR8) and the other by continuous exposure to cisplatin (RT112-CP). RT112-DXR8 cells were 1.6- to 2-fold more sensitive to cisplatin and 2 analogues, carboplatin and iproplatin, compared with the parental line, whereas RT112-CP cells were 1.6- to 2.8-fold more resistant to these agents. Uptake of 195mcisplatin was elevated 1.4-fold in RT112-DXR8 cells compared with RT112-P cells whereas uptake into RT112-CP cells was similar to that of the parental line. Binding of 195mcisplatin to DNA was similar in all 3 lines. Levels of reduced glutathione were significantly elevated in RT112-CP cells and significantly reduced in RT112-DXR8 cells compared with the parental cells. In addition, activities of glutathione reductase and glutathione peroxidase were higher in RT112-CP cells than in the parental cells whereas the activity of glutathione-S-transferase was similar in all 3 cell lines. A 2.5-fold greater induction of DNA-DNA interstrand crosslinks occurred in RT112-DXR8 cells compared with the parental line, whereas crosslinking in RT112-CP cells, whilst initially similar to that seen in RT112-P cells, was significantly elevated at later times. These findings suggest that mechanisms associated with the expression of resistance and collateral sensitivity to cisplatin may differ.

Cell Line↗
PubMed1987

Intravesical chemotherapy: combination with Tween 80 increases cytotoxicity in vitro.

Tween 80 was shown to enhance significantly the cytotoxic activities of the four drugs (adriamycin, epodyl, mitomycin-c, thiotepa) most frequently administered intravesically to treat superficial bladder cancer. The colony forming ability of a human bladder cancer cell line, RT112, was measured following a 1 h exposure to each of the four drugs both alone and in combination with 0.1% and 0.3% Tween 80. Cell survival was not reduced by 0.1% Tween 80 alone. We conclude that the combination of Tween 80 with these drugs might increase the therapeutic index of intravesical chemotherapy.

Administration, Intravesical↗
PubMed1987

Factors influencing the sensitivity of two human bladder carcinoma cell lines to cis-diamminedichloroplatinum(II).

A two-fold difference in sensitivity to cis-diamminedichloroplatinum(II) (cisplatin), as judged by colony forming assays, has been demonstrated in two human bladder carcinoma continuous cell lines. Approximately twice as many DNA-DNA interstrand cross-links (ISL) and a 2-fold greater inhibition of DNA synthesis occurred in the more sensitive T24 cell line than in the RT112 cell line after exposure to the same concentrations of cisplatin. Equitoxic concentrations of cisplatin resulted in similar extents of ISL and inhibition of DNA synthesis in both cell lines. Although drug uptake was identical, twice as much cisplatin was bound to the DNA of T24 cells than RT112 cells. However after equitoxic concentrations of cisplatin the DNA from both cell lines was platinated to a similar extent. In addition, levels of glutathione (GSH), glutathione reductase (GR) and total glutathione-S-transferases (GST) were higher in the less sensitive RT112 cell line.

Biological Transport↗
PubMed1987

Affinity purification of a high molecular weight human breast cancer-associated antigen identified by the BCD-B4 monoclonal antibody.

This study reports the purification and characterization of a high molecular weight human breast cancer-associated antigen identified by a previously described (1,2) murine monoclonal antibody, BCD-B4. Immunohistochemical analysis indicated that BCD-B4 recognizes an antigen expressed in an altered form on the human breast carcinoma cell line, BT-20, compared to the non-malignant human mammary epithelial cell line, HBL-100. Chemical treatments and enzymatic digestions suggested that the recognized moiety was a protein. The antigenic determinant was resistant to neuraminidase and periodate treatments but was sensitive to trypsin and proteinase K. The antigen was purified by affinity chromatography and its molecular weight, determined by SDS-PAGE analysis under non-reducing conditions, was proven to be 250 Kd. Under reducing conditions, the molecule dissociated into two polypeptides of 125 and 45 Kd, respectively. Both subunits could be isolated from normal HBL-100 and neoplastic BT-20 cellular protein extracts by affinity chromatography. The higher molecular weight subunit showed; however, qualitative and quantitative differences between the two cell lines: it was expressed in greater quantity on BT-20 cells and its molecular weight was 15 Kd higher. Both subunits could also be identified by immunoblots of BT-20 cells.

Antibodies, Monoclonal↗
PubMed1987

Differential sensitivities of human testicular and bladder tumor cell lines to chemotherapeutic drugs.

The in vitro drug sensitivities of 5 human testicular tumor cell lines (Tera II, SuSa, NEC-8, 833K, T3B1) and 5 human bladder carcinoma cell lines (RT4, RT112, T24, HT1197, HT1376) were compared. Cytotoxicities of cisplatin and doxorubicin were assessed by inhibition of colony-forming ability during continuous exposure to a range of drug concentrations. The ranges of the drug concentrations required to kill 70% of clonogenic cells obtained against the testicular cell lines were 1-7 ng/ml and 21-161 ng/ml for doxorubicin and cisplatin, respectively, compared with 4-19 ng/ml and 112-431 ng/ml for the bladder cell lines. This study shows that continuous cell lines retain the relative clinical chemosensitivities of their tumors of origin. The results also indicate that testicular tumor cells are inherently more sensitive to the cytotoxic effects of chemotherapeutic drugs than are bladder cancer cells.

Cell Line↗
PubMed1987

The origin and movement of gas during adult emergence in Aedes aegypti: an hypothesis.

Several events which precede adult emergence in Aedes aegypti were studied, including changes in buoyancy, changes visible through the pupal cuticle and changes at the foregut-midgut junction. Our data suggest tht the gas which is present in the posterior midgut at the time of emergence originates in the tracheal system. During the process of emergence this gas moves into the exuvial space through the adult spiracles and then follows the exuvial fluid into the alimentary canal.

Aedes↗