Search PubMed⌕ Search

Biomedical subjects

M Melamed

Publications and source records attributed to M Melamed.

At least 19 recordsLinked to original sources
PubMed1997

Cellular changes and induction of apoptosis in human promyelocytic HL-60 cells infected with the agent of human granulocytic ehrlichiosis (HGE).

Human granulocytic ehrlichiosis (HGE) is an emerging and occasionally fatal human infectious disease whose pathogenesis is largely unknown. Goodman et al. (1) recently described the successful cultivation of the HGE infectious agent in human promyelocytic HL-60 leukemic cells. It was reported in the same study that infectivity invariably led to host cell death, although the mechanism by which HGE infection triggers cellular self-destruction is as yet undetermined. In this communication, we show that in vitro passage of HGE pathogen-infected blood elicits a significantly dysfunctional G1-to-S transition. Moreover, we provide evidence that the cytopathic properties of the HGE pathogen are attributed to its ability to induce apoptosis in host HL-60 cells. Determination of specific protein expression changes by Western blot analysis showed that HGE infection resulted in reduced expression of PCNA and pRB, both of which play a role in cell cycling. Moreover, the steady state level of bcl-2, which protects eukaryotic cells against apoptosis, is suppressed by exposure to the HGE agent. These results suggest that this pathogen HGE induces apoptosis in HL-60 cells by a mechanism involving the shut-off of multiple cell cycle and apoptosis regulatory events.

Apoptosis↗
PubMed1997

Estradiol-binding mechanism and binding capacity of the human estrogen receptor is regulated by tyrosine phosphorylation.

We have investigated the effects of tyrosine phosphorylation on the estradiol-binding mechanism and binding capacity of the human estrogen receptor (hER). The wild type hER and a point mutant form of the hER, in which tyrosine 537 was mutated to phenylalanine (Y537F hER), were expressed in Sf9 insect cells. The wild type hER, but not the Y537F hER, reacted with a anti-phosphotyrosine monoclonal antibody, indicating that tyrosine 537 was the only tyrosine phosphorylated on the hER. Scatchard and Hill analyses of the the binding interaction of [3H]estradiol with the wild type hER indicated that the addition of millimolar phosphotyrosine, but not tyrosine, phosphate, or phosphoserine, abolished the cooperative binding mechanism of the hER. These observations are consistent with the idea that phosphotyrosine blocks dimerization and site-site interactions between the hER monomers. The wild type hER bound 10-fold more [3H]estradiol than the Y537F hER. Treatment of the purified wild type hER with a tyrosine phosphatase decreased the binding capacity of the hER by approximately 90%, whereas, a serine/threonine phosphatase had no effect. The estrogen-binding capacity of the tyrosine-dephosphorylated hER was completely restored by rephosphorylation of tyrosine 537 with p60c-src, a tyrosine kinase. These results indicate that p60c-src can restore estrogen binding to the tyrosine-dephosphorylated hER and that dimerization and cooperative site-site interaction of the hER occur via a phosphotyrosine-binding interaction.

Dimerization↗
PubMed1997

Molecular and kinetic basis for the mixed agonist/antagonist activity of estriol.

Estriol acts as a weak estrogen when administered in a single dose into immature or ovariectomized laboratory animals, but produces full estrogenic responses upon chronic administration. However, when estriol is injected together with estradiol it acts as an antiestrogen. We studied the dual agonist/antagonist properties of estriol, using recombinant human estrogen receptor (hER) in ligand-binding assay, cell-free transcription assay, electrophoretic mobility shift assay with cVitII estrogen response element (ERE), and ERE-Sepharose chromatography. We show that the weak estrogenic activity of estriol results from impaired hER-ERE interaction. The antiestrogenic activity of estriol was demonstrated in a cell-free transcription assay where it reduced estradiol-dependent transcription in a dose-dependent manner. Estriol interfered with estradiol-induced positive cooperative binding and receptor dimerization, and binding of hER complexes to ERE. These effects of estriol were maximal at a 10-fold molar excess over estradiol; under these conditions estradiol-dependent transcription was decreased by 85%, although [3H]estradiol binding was reduced by only 50%. We propose that when hER, estradiol, and estriol are coequilibrated, several receptor species are formed: unliganded hER monomers and dimers; estradiol-hER monomers and dimers, estriol-hER monomers and dimers; and presumably mixed estradiol-estriol dimers. Since estrogen-hER complexes bind cooperatively to ERE sequences, the concentrations of transcriptionally active complexes (estriol- and estradiol-hER dimers) are reduced to low levels that fail to bind cooperatively with ERE and initiate transcription. We discuss our results in relation to the massive estriol production during pregnancy and to the "Estriol Hypothesis" on the protective role for estriol in opposing carcinogenic effects of estradiol.

Cell-Free System↗
PubMed1996

Kinetic analysis of the interaction of human estrogen receptor with an estrogen response element.

The kinetics of the interaction between recombinant human estrogen receptor and chicken vitellogenin gene II estrogen response element (ERE) were determined by ERE-Sepharose chromatography. The association constant of the interaction between the ERE and the human estrogen receptor was dependent on receptor concentration, estradiol binding and temperature. The highest association constant (80-100 x 10(6)M-1) was measured for the estradiol-bound receptor prepared at 25 degrees C and at concentrations higher than 7 nM. At high receptor concentrations (>7 nM) the binding mechanism of estradiol to the receptor was positive cooperative, indicating receptor homodimerization. At lower concentrations the binding mechanism was partially cooperative and the association constant of the liganded receptor was significantly lower. The binding mechanism at 4 degrees C was cooperative as well, and the association constants were similarly dependent upon receptor concentration, but were 50% lower than the receptor prepared at 25 degrees C. The association constant of the unliganded receptor was 4- to 5-fold lower than that of the liganded receptor at 25 degrees C. These data suggest that in addition to estradiol-induced conformational changes in the receptor, the receptor dimers are subjected to temperature-dependent changes, which further increase their affinity for an ERE.

Animals↗
PubMed1994

In vitro bromodeoxyuridine labeling of malignant neoplasms. A comparative study with flow cytometry cell-cycle analysis.

Proliferative fraction has been defined as an independent prognostic marker for some malignant neoplasms. An estimate of the proliferative fraction can be obtained by cell-cycle analysis of flow cytometric DNA measurements. However, overlapping DNA distributions of aneuploid neoplasms are difficult to analyze, even with sophisticated computer programs, and results are not always reproducible. Bromodeoxyuridine (BrdUrd) labeling followed by immunohistochemical detection has been proposed as an alternative, simple, and accurate method for identifying and counting DNA synthesizing cells. In vitro BrdUrd labeling was performed on 87 tumors, including 35 lung cancers, 25 breast carcinomas, and 27 tumors of other origin. Results were compared with flow cytometric S-phase estimates in 46 cases. Mean BrdUrd labeling of lung tumors was 8.5% +/- 5.2%, compared with a mean flow cytometric S-phase fraction of 20% +/- 18.4%. Mean BrdUrd labeling of breast carcinomas was 6.8% +/- 3.8%, compared with a mean flow cytometric S-phase fraction of 12.5% +/- 9.9%. In both tumor types, BrdUrd labeling correlated well with histologic grade. Correlation between BrdUrd labeling and flow cytometric S-phase estimates were generally poor, particularly with aneuploid tumors.

Breast Neoplasms↗
PubMed1993

Hypocellular bladder wash specimens and their clinical significance.

Bladder wash flow cytometry specimens containing less than 1,000 cells were defined as hypocellular. Between June 1981 and December 1990, 5,676 bladder wash flow cytometry specimens were collected, of which 142 (2.5%) were deemed hypocellular. Of the 132 evaluable patients with hypocellular specimens 34 (30%) had documented evidence of carcinoma compared to 52% for the entire population. Among the 34 patients 30 had transitional cell carcinoma, of which 12 were carcinoma in situ, 9 stage Ta or T1 and 9 stage T2 or greater. Thus, there was no obvious relationship between stage or grade of carcinoma versus probability of a hypocellular specimen. Differences in irrigation techniques of different urologists in the operating room using general anesthesia versus catheter and cystoscopic irrigation in the outpatient clinic are discussed as possible explanations for the hypocellular specimens.

Biopsy↗
PubMed1991

Human monoclonal antibodies to human blood group antigens Kidd Jka and Jkb.

Three IgM human monoclonal antibodies to Jkb, and one IgM human monoclonal antibody to Jka were produced from the lymphocytes of two immunized donors. Two of the anti-Jkb monoclonal antibodies (MS-7 and MS-9) are of the IgM (kappa) isotype and one (MS-8) is an IgM(lambda). The anti-Jka monoclonal antibody (MS-15) is of the IgM(kappa) isotype. They are all specific for their respective antigens, and give positive agglutinations in saline by the immediate spin technique, even against Jk(a+b+) cells. The heterohybridomas have been shown to be suitable for bulk culture and produce levels of antibody that reach 18 micrograms/ml in the spent culture supernatant. They offer considerable advantages over currently available reagents in terms of stability, simplicity and speed of use, and will provide a reliable and unlimited supply of what are at the moment rare and unsatisfactory antibodies.

Antibodies, Monoclonal↗
PubMed1990

Human monoclonal antibodies to C, c, E, e and G antigens of the Rh system.

A panel of heterohybridomas secreting human IgG and IgM monoclonal antibodies to the C, c, G, E, and e antigens of the Rh system has been established. Both classes of antibody have been shown to react with red blood cells carrying their respective antigens; those of the IgM class being able to directly agglutinate unmodified red blood cells in saline. The heterohybridomas have been shown to be suitable for bulk culture and produce levels of antibody in the range of 15-73 micrograms/ml in the spent culture supernatant.

Antibodies, Monoclonal↗
PubMed1989

Flow cytometry as a predictive indicator in patients with operable gastric cancer.

Adenocarcinoma of the proximal portion of the stomach (gastroesophageal [GE] junction and cardia) is increasing in incidence. The inferior survival of patients with GE-cardia lesions as compared with patients with tumors located in the body and antrum has been attributed to anatomic features. To determine if a biological difference could explain the varying prognosis, flow cytometric studies were performed prospectively in 50 patients with operable gastric cancer and analyzed for association with site, histology, gender, age, stage, and disease-free survival. DNA aneuploidy significantly correlated with tumor location: 96% of GE-cardia carcinomas were aneuploid as compared with 48% of body-antrum tumors (P = .0008). Nodal involvement was more common in aneuploid tumors (P = .0548), and women were more likely to have diploid tumors than were men (P = .0233). The median disease-free survival for patients with diploid tumors was 18.5 months as compared with 5.4 months for patients with aneuploid carcinomas (P = .076). Furthermore, within the body-antrum of the stomach, patients with diploid tumors had a significantly better disease-free survival than did those with aneuploid tumors from the same site (18.4 v 4.7 months, P = .0185). These results indicate there is a difference in the DNA content of gastric tumors located in different sites within the stomach and that DNA content correlates with prognosis.

Adenocarcinoma↗
PubMed1987

Influence of tumor cell DNA ploidy on the natural history of rectal cancer.

We have examined tumor cell DNA content as a possible variable in the behavior of early rectal cancer treated by local excision. Flow cytometry assays of tumor cell DNA content were carried out on specimens of archived, paraffin-embedded tissue specimens from 30 patients (11 male and 19 female) whose early rectal cancers were treated by curative local excision more than 60 months previously. The cancers invaded to the muscularis mucosae in 2 patients (1 with aneuploid disease and 1 with diploid disease), into the submucosa in 15 patients (7 with aneuploid disease and 8 with diploid disease), and the muscularis propria in 13 patients (8 with aneuploid disease and 5 with diploid disease). A total of 16 patients had aneuploid disease and 14 had diploid disease. Local recurrence of cancer developed in 12 patients. Of these 12 patients, 10 (83 percent) had aneuploid disease. By contrast, of the 18 patients who remained free of disease, 12 (67 percent) had diploid disease. Seven of the 12 patients with recurrence died. Six of these seven (86 percent) had aneuploid disease. The aggressive clinical behavior of the tumors with aneuploid DNA content was not otherwise predictable by standard histologic features. Aggressive tumor behavior appears to correlate closely with aneuploidy in locally treated rectal cancers, as opposed to a lack of correlation in our patients treated with major resection. The fact that these cancers are being treated by local excision may allow the prognostic impact of DNA content to reflect the natural history of cancer.

Adenocarcinoma↗
PubMed1987

beta-Glucosidase in Candida albicans and its application in yeast identification.

In this report we attempt to explain the discrepancy between beta-glucosidase (EC 3.2.1.21) activity in Candida albicans as measured by commercial kits and that found in an experimental assay. beta-Glucosidase activity in American and Israeli isolates of C. albicans was evaluated with the API ZYM and YeastIdent systems (Analytab Products) and with experimental biochemical assays. Activity was found with whole cells and cell extracts of isolates from both sources. The greatest beta-glucosidase activity was found at pH 5.0 and with p-nitrophenyl-beta-glucopyranoside (PNP-BDG) as the substrate. In assays with beta-naphthyl-beta-D-glucopyranoside and 6-bromo-2-naphthyl-beta-D-glucopyranoside (6-Br-2-naphthyl-BDG), no enzyme activity was detected in whole cells and only limited activity was found in cell extracts of isolates from both sources. In studies with PNP-BDG at pH 5.0 and 7.5, 29 to 38% less activity was found at both pHs with American whole cells, and minor activity (20%) was found at pH 7.5 with isolates from both sources. Because assays with PNP-BDG in cell extracts of isolates from both sources showed no significant differences in activity, the more limited beta-glucosidase activity in American whole cells was most likely due to less efficient transport. Because the API ZYM system uses 6-Br-2-naphthyl-BDG as the substrate and because the substrate is buffered at pH 7.5 in the API YeastIdent kit, both systems appear to be of limited value for the detection of beta-glucosidase activity in C. albicans.

Candida albicans↗
PubMed1986

Phase I/II trial of intravesical methotrexate for superficial bladder tumors.

Twenty-one patients with superficial transitional cell carcinoma of the bladder received a total of 121 doses of intravesical methotrexate (MTX) at 11 different concentrations of drug, ranging from 40 mg/m2 (mean concentration of 2.9 X 10(-3) M) to 500 mg/m2 (4.9 X 10(-2) M). Biochemical evidence of absorption was minimal in all cases. The maximum serum level was observed within 0.5-2 h in all patients and ranged from 1.8 X 10(-8) M to 5.0 X 10(-7) M. By 24 h the serum levels were negligible and ranged from 5.5 X 10(-9) M (the lowest limit detectable by the assay) to 4.4 X 10(-8) M in the patient who received the highest dosage of 500 mg/m2. Biologic evidence of absorption was minimal. Myelosuppression, mucositis, and nausea were not observed. Eighteen patients received six consecutive weekly doses ranging from 40 to 500 mg/m2. All patients had repeat cytoscopy performed within 2-4 weeks after six consecutive doses to evaluate local toxicity and efficacy. Flow cytometry was performed on the bladder washings of 22 patients, illustrating the use of flow cytometry, in conjunction with conventional cytology, as an additional means of objectively quantifying results. Despite MTX's established activity in systemic treatment of advanced bladder carcinoma, this study failed to demonstrate any clinical response to intravesically administered MTX, in doses of up to 500 mg/m2, and in concentrations of up to 4.9 X 10(-2) M.

Adult↗
PubMed1985

Investigations of serum testosterone levels in patients with laryngeal cancers.

Serum testosterone levels were determined by a radioimmunological method in 102 patients with laryngeal carcinoma and in a control group of 10 healthy men. All patients were examined before the initiation of any operative or radiation therapy and after termination of treatment. The testosterone levels in the cancer patients showed a tendency toward higher values when compared with a control group of healthy men. These results indicate that hormone preparations could be considered as part of the therapy of laryngeal carcinomas.

Adult↗
PubMed1985

Immunohistologic dissection of the human kidney using monoclonal antibodies.

Nine murine monoclonal antibodies which detect differentiation antigens of the human kidney are described. Immunofluorescence and immunoperoxidase studies demonstrate that these antigens are expressed by different cell types comprising the nephron. Monoclonal antibody MA99 detects a glycoprotein complex of the glomerular basement membrane. Monoclonal antibody S4 detects a glycoprotein of 160,000 daltons (gp160) expressed by glomerular and proximal tubular epithelial cells. Monoclonal antibodies S23, S27 and S6 immunoprecipitate a glycoprotein of 120,000 daltons (gp120) found on cells of the proximal tubule and portions of Henle's loop. Monoclonal antibody C26 identifies a glycoprotein of 40,000 daltons (gp40) expressed by cells of the distal and collecting tubules. Monoclonal antibodies M2 and S8 are specific for A and B blood group antigens, respectively, found on cells of the collecting tubule in individuals of the respective blood type. This panel of antibodies is useful in the study of normal renal embryogenesis, microanatomy and physiology as well as pathological processes including tumors.

Animals↗
PubMed1984

Recognition of central nervous system leukemia by flow cytometry.

Cerebrospinal fluid of 24 patients with acute leukemia was studied by DNA/RNA flow cytometry. In six of 15 patients with central nervous system (CNS) relapse, the spinal fluid cells had abnormal DNA stemlines, ranging from near haploid to hyperdiploid. In two additional cases, leukemic cells were identified by a abnormally high RNA content only. One patient had two different aneuploid cell populations in spinal fluid not distinguished by cytologic morphology. Another patient with initial diploid leukemia had CNS relapse characterized by the same DNA stemline long after a triploid DNA stemline emerged in the marrow. DNA/RNA flow cytometry detected leukemic cells that were not identified ("uniform" or "reactive") by cytological criteria in 5/6 patients studied and in addition differentiated lymphoblastic from nonlymphoblastic cell types according to low and high RNA content.

DNA, Neoplasm↗