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Biomedical subjects

D Greene

Publications and source records attributed to D Greene.

At least 37 records · Page 2Linked to original sources
PubMed1990

Affinity purification of tissue plasminogen activator using transition-state analogues.

The search for a simple affinity ligand to purify tissue plasminogen activator (tPA) was facilitated by a solid-phase synthesis approach. A large variety of tripeptide ligands containing argininal were synthesized on agarose gels containing a spacer with carboxy terminal. The immobilized ligands were easy to test with urokinase, and tPA. While a number of sequence combinations showed initial binding by tPA, only a few resulted in tight binding corresponding to a hemiacetal linkage with the active site serine. Hydrophobic residues, especially aromatics, flanking the N-side of argininal gave rise to ligands which were bound strongly by tPA. A gel containing D-Phe-D-Phe-Argal (an aldehyde derivative of arginine) was very effective in purifying tPA derived from cell culture media at small scale (milligrams) and at large (multi-grams).

Amino Acid Sequence↗
PubMed1990

Effects of the ACAT inhibitor CL 277,082 on cholesterol metabolism in humans.

A common pharmacologic approach to lowering elevated serum cholesterol levels has been to interfere with intestinal sterol absorption. Inhibitors of acyl coenzyme A: cholesterol acyltransferase (ACAT) should produce this effect. In this study, we examined the effects of CL 277,082, N-(2,4-difluorophenyl)-N-(4-neopentylbenzyl)-N-(n-heptyl)urea, an ACAT inhibitor, on cholesterol metabolism in humans. Eight healthy male volunteers were given a placebo for 14 days, followed by 750 mg/day CL 277,082 for 20 days in a single-blind, crossover design. Subjects were studied in a hospital research unit and were fed strictly controlled diets. Cholesterol absorption was measured by the dual isotope method during the final week of both the placebo and the drug phases. Sterol balance was also assessed during these two periods by measuring cholesterol intake, and fecal neutral and acidic sterol excretion rates. Plasma lipids and lipoproteins were measured at the end of each period. The drug was well tolerated and produced no detectable clinical or laboratory side effects. Cholesterol absorption, sterol excretion rates, and plasma lipoprotein levels were all unaffected by treatment. We conclude that CL 277,082 may not interfere with ACAT activity or cholesterol absorption in humans at the doses given under the conditions tested in this study.

Adult↗
PubMed1990

Radiation response of drug-resistant variants of a human breast cancer cell line: the effect of glutathione depletion.

Two drug-resistant variants of the human breast cancer cell line MCF-7 have been shown previously to exhibit radiation resistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, glutathione (GSH) depletion was achieved by exposure of cells to buthionine sulfoximine (BSO) with, in some cases, additional treatment with dimethyl fumarate. Levels of GSH in the adriamycin-resistant subline MCF-7 ADRR are initially lower than in the other two sublines and are depleted to a greater extent by exposure to BSO. Wild-type MCF-7 cells are not sensitized by GSH depletion when irradiated under aerated conditions but are sensitized under hypoxic conditions to an extent which is related to the level of GSH depletion. In contrast both the drug-resistant sublines (MCF-7 ADRR and the melphalan-resistant line MCF-7 MLNR) are radiosensitized by GSH depletion under both aerated and hypoxic conditions. It is hypothesized that in the case of the MCF-7 ADRR cell line, which expresses high levels of the GSH-associated redox enzyme systems, GSH-S-transferase and GSH-peroxidase (GSH-Px), radiosensitization results when GSH-Px is inhibited in GSH-depleted cells. The reasons for radiosensitization of aerated MCF-7 MLNR cells cannot be explained on this basis, however, and other factors are being examined.

Antineoplastic Agents↗
PubMed1989

Radiation response of drug-resistant variants of a human breast cancer cell line.

The radiation response of drug-resistant variants of the human tumor breast cancer cell line MCF-7 has been investigated. Two sublines, one resistant to adriamycin (ADRR) and the other to melphalan (MLNR), have been selected by exposure to stepwise increasing concentrations of the respective drugs. ADRR cells are 200-fold resistant to adriamycin and cross-resistant to a number of other drugs and are characterized by the presence of elevated levels of selenium-dependent glutathione peroxidase and glutathione-S-transferase. MLNR cells are fourfold resistant to melphalan and cross-resistant to some other drugs. The only mechanism of drug resistance established for MLNR cells to date is an enhancement of DNA excision repair processes. While the spectrum of drug resistance and the underlying mechanisms differ for the two sublines, their response to radiation is qualitatively similar. Radiation survival curves for ADRR and MLNR cells differ from that for wild-type cells in a complex manner with, for the linear-quadratic model, a decrease in the size of alpha and an increase in the size of beta. There is a concomitant decrease in the size of the alpha/beta ratio which is greater for ADRR cells than for MLNR cells. Analysis of results using the multitarget model gave values of D0 of 1.48, 1.43, and 1.67 Gy for MCF-7 cells are not a consequence of cell kinetic differences between these sublines. Results of split-dose experiments indicated that for both drug-resistant sublines the extent of sublethal damage repair reflected the width of the shoulder on the single-dose survival curve. For MCF-7 cells in the stationary phase of growth, the drug-resistant sublines did not show cross-resistance to radiation; however, delayed subculture following irradiation of stationary-phase cultures increased survival to a greater extent for ADRR and MLNR cells than for wild-type cells.

Antineoplastic Agents↗
PubMed1989

Enhanced DNA cross-link removal: the apparent mechanism of resistance in a clinically relevant melphalan-resistant human breast cancer cell line.

Resistance to the cytotoxic effects of alkylating agents is a major limitation to their clinical efficacy. Although a number of animal and human tumor cell models have been developed to study this problem, it has proven difficult to achieve very high levels of resistance to alkylating agents in vitro. This is consistent with the recent clinical evidence that alkylator resistance can be overcome by dose escalations of less than 10-fold. A number of mechanisms of alkylator resistance have been described, more than one of which may occur in the same model. This paper describes a human breast cancer cell subline selected for 3-fold resistance to melphalan and cross-resistant to other alkylators in which only one of the previously described mechanisms of resistance, enhanced removal of DNA interstrand cross-linking, is demonstrable. Northern blot analysis using the human incisional repair gene ERCC-1 cDNA demonstrated that this particular gene product is not the altered function in these cells, so the molecular characterization of the observed enhanced repair is pending. Because these cells are also cross-resistant to radiation and to adriamycin and epipodophyllotoxin, they may represent a clinically relevant model in which to examine the role of DNA repair of lesions resulting from alkylators and other cytotoxic agents.

Blotting, Northern↗
PubMed1988

The pathogenesis and prevention of diabetic neuropathy and nephropathy.

The chronic complications of diabetes are thought to be caused by an interaction between hyperglycemia, or other metabolic consequences of insulin deficiency, and independent genetic or environmental factors that are poorly defined. Several potentially relevant biochemical sequelae to hyperglycemia have been identified in tissue susceptible to diabetic complications. Among these, a rise in tissue sorbitol secondary to concentration-dependent activation of polyol pathway activity by glucose, and an accompanying fall in tissue myo-inositol and Na-K-ATPase activity have recently been linked to a self-reinforcing cyclic metabolic defect that accounts for rapidly reversible slowing of conduction in peripheral nerve in diabetes. Impaired Na-K-ATPase activity also appears to be responsible for intracellular Na+ accumulation and resultant localized axonal paranodal swelling that characterizes diabetic neuropathy in both humans and laboratory animals. These swellings are thought to be responsible for the subsequent disruption of the nodal apparatus (axo-glial disjunction) and some component of the loss of large and small myelinated fibers. Recent studies have suggested that microvascular insufficiency may also contribute to diabetic neuropathy, especially in non-insulin-dependent diabetes. Aldose reductase activity is concentrated in endoneurial vessels, and similar biochemical mechanisms (ie, sorbitol accumulation, myo-inositol deficiency, and impaired Na-K-ATPase activity) are thought to be operative in the endoneurial microvessels in diabetes. Administration of an aldose reductase inhibitor to patients with diabetic neuropathy is associated with repair of damaged nerve fibers and the appearance of newly generated fibers, presumably secondary to metabolic correction within the nerve fibers themselves or their supporting microvasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗
PubMed1986

Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component.

Yeast killer toxin and a component giving immunity to it are both encoded by a gene specifying a single 35 kd precursor polypeptide. This precursor is composed of a leader peptide, the alpha and beta subunits of the secreted toxin, and a glycosylated gamma peptide separating the latter. The toxin subunits are proteolytically processed from the precursor during toxin secretion. Using site-directed mutagenesis, we have identified a region of the precursor gene necessary for expression of the immunity phenotype. This immunity-coding region extends through the C-terminal half of the alpha subunit into the N-terminal part of the gamma glycopeptide. Mutations in other parts of the gene allow full immunity but produce precursors that fail to be processed. The precursor can therefore confer immunity, and we propose that it does so in the wild type by competing with mature toxin for binding to a membrane receptor.

Fungal Proteins↗
PubMed1985

A randomised comparison of photons and 15 MeV neutrons for the treatment of carcinoma of the bladder.

108 patients with T2 or T3 carcinoma of the bladder were randomised to receive either photon therapy or low- or high-dose 15 MeV neutrons. Fifty-nine patients received photon treatment, 20 low-dose neutrons (LDN) and 20 high-dose neutrons (HDN). Eight patients received a combination of photon and neutron therapy, due to machine breakdown, and one patient received only one exposure, due to intercurrent illness. The three-year survival for all groups was 43% but there was no difference in survival between the treatment groups. The complication rate in the photon group was low--minor 3%, major 5%. The complication rates in the neutron groups were higher--LDN 30% and 15% respectively, HDN 15% and 10% and combined 25% and 37%. Only six patients died of complications related to therapy--photons 2, LDN 1, HDN 1, combined 2. No therapeutic gain for neutron therapy has been found in this study.

Clinical Trials as Topic↗
PubMed1984

Isolated adrenal masses in nonsmall-cell bronchogenic carcinoma.

Computed tomography has become an important diagnostic modality in the preoperative staging of patients with bronchogenic carcinoma. The adrenal glands represent one of the most frequent sites of metastasis. Therefore, an isolated adrenal mass discovered on preoperative thoracoabdominal CT poses a diagnostic problem. Three hundred thirty patients with histologically proved nonsmall-cell bronchogenic carcinoma were evaluated. Thirty-two had adrenal masses without further evidence of disease in the abdomen. Eight of these 32 masses were metastases, 17 were proved adenomas, and seven did not undergo biopsy. Thus, in the patients with nonsmall-cell bronchogenic carcinoma, an isolated adrenal mass is more likely benign than metastatic, and biopsy is advocated prior to withholding potentially curative surgery.

Adolescent↗
PubMed1983

Secretion of Saccharomyces cerevisiae killer toxin: processing of the glycosylated precursor.

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2. Protoxin stability in kex1 kex2 double mutants indicated the order kex1 --> kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1- and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

Killer Factors, Yeast↗