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Biomedical subjects

G Quash

Publications and source records attributed to G Quash.

At least 37 records · Page 2Linked to original sources
PubMed1992

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.

Animals↗
PubMed1991

Differential expression of isopeptide bonds N epsilon (gamma-glutamyl) lysine in benign and malignant human breast lesions: an immunohistochemical study.

N epsilon (gamma-glutamyl) lysine isopeptide bonds were detected in situ in histological sections from benign and malignant human breast tissue using the monoclonal antibody (MAb) 81D1c2. On cryostat sections of fresh-frozen mammary tissue post-fixed in acetone, or on paraffin sections also from mammary tissue fixed in Bouin's solution, the MAb reacted preferentially with glandular epithelial cells and staining was restricted to the nuclei. In 41 out of 44 benign lesions examined, staining was strong or moderate, while in 25 out of 33 malignant lesions no staining was observed. In the 8 remaining lesions of this group, staining was positive but weak. The difference in reactivity of this MAb with the 2 types of lesions is highly significant (p less than 0.0005) according to the chi 2 test.

Adult↗
PubMed1991

Transglutaminase activity and N epsilon (gamma glutamyl) lysine isopeptide levels during cell growth: an enzymic and immunological study.

A monoclonal antibody (MAb) 81D1c2, which recognizes the N epsilon (gamma glutamyl) lysine isopeptide produced by the action of transglutaminase activity was prepared. Its reactivity towards the homologous isopeptide was about 3-fold greater than that with either N alpha (alpha glutamyl) lysine (a naturally occurring heterologous dipeptide) or N alpha (gamma glutamyl) lysine, another heterologous peptide not described so far in naturally occurring proteins. When used in an immunohistochemical study on cells in culture derived from human carcinoma of the larynx (HEp2) and from chicken embryo cells (CEC), both fixed in acetone, this MAb detected N epsilon (gamma glutamyl) lysine residues in the nucleus. The amount of N epsilon (gamma glutamyl) lysine isopeptides follows closely transglutaminase activity during the lag phase of growth of both CEC and HEp2 cells. However, during exponential growth, the 2 parameters decrease concomitantly in HEp2 cells, whereas in CEC, transglutaminase activity increases but isopeptide bond levels drop. Compared with other reported methods for measuring isopeptides, this immunohistological approach permits the localization and at least the semi-quantitative determination of N epsilon (gamma glutamyl) lysine in cells in situ.

Animals↗
PubMed1991

Epidemiology of measles in the Cameroons between 1984 and 1986: comparison of the effectiveness of different serological methods in rural regions.

Epidemiological studies were carried out in Yaoundé and in Ngaoundéré from 1984 to 1986, in an attempt to develop adequate methods of collecting blood from small children and of diagnosing measles appropriate to conditions in the field. Alternative methods were necessary since classical methods used in modern laboratories are unsuitable in rural regions. Each study was carried out on a representative sample of 6- to 36-months-old infected children seen at consultation. This group was chosen because it suffers the highest mortality rate. The blood was obtained by digital puncture on blotting paper because venepuncture required sterile equipment and also the establishment of a cold chain for transporting the samples to the laboratory. The first criteria examined were the relative titers of sera taken by finger prick. Four serological techniques were used: indirect immunofluorescence (IIF), indirect immunoperoxidase (IIP), hemagglutination inhibition (HAI) and ELISA. Antimeasles antibodies were detected in 12% of infected children using IIF and in 18% using IIP. When sera were examined by HAI the percentage of positives was 54% and by ELISA 75%. These results clearly indicate that ELISA is the most effective and practical technique for diagnosing measles under field conditions.

Antibodies, Viral↗
PubMed1991

Purification of measles virus by affinity chromatography and by ultracentrifugation: a comparative study.

The purification of viruses by ultracentrifugation in regions with limited resources is often hampered by problems of instrument maintenance. Viral antigens must therefore be imported, which results in delays in obtaining epidemiological data. To address this problem, we undertook the development of purification procedures by affinity chromatography using measles virus (strain Y15) and monkey IgG (anti-measles) coupled covalently via their carbohydrate residues to glass spheres as a model system. The measles virus prepared in this way was compared with measles virus purified by ultracentrifugation using three criteria: the degree of purification, yield and the sensitivity in HA and ELISA. The purification of the antigen by ultracentrifugation (AgU) was undertaken using 10(9) infected cells, and by affinity chromatography (AgAc) using 10(7) infected cells. The total concentration of proteins before purification was 5000 mg for AgU and 11.3 mg for AgAc. After purification by AgU 21.5 mg protein was obtained which was assessed by specific hemagglutinating units (HAU/mg protein) and found to be 179 for AgU and 372-744 for AgAc. Based on total HAU, the yield by centrifugation was 2.4%, whereas the affinity chromatography produced essentially 100%. By ELISA, using reference sera to calibrate the antigens, maximum OD was obtained with 0.5 microgram/well of AgAc and with 2 micrograms/well of AgU. From the relatively small amount of starting material, good yields were obtained with increased specific activity economy in coating by ELISA. These results favour the adoption of affinity chromatography for purifying viral antigens.

Animals↗
PubMed1990

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies of defined specificity. I. Application to naturally occurring antipolyamine antibodies in human sera.

A simple covalent enzyme-linked immunoassay procedure (CELIA) is described for the routine determination of free and immune complex-bound antibodies in sera. Assays for the latter could not have been performed by adsorption ELISA due to the high ionic strength of the reassociating buffer. For the measurement in human sera of free naturally occurring IgG and IgM antibody directed against the hapten spermine, polycarboxystyrene microtiter plates with covalently coupled spermine were used. For the determination of immune complex-bound antipolyamine IgG and IgM antibody titers, serum was first dissociated at pH 2.3 in tubes and then reassociated at pH 8.1 in the wells of a microtiter plate containing covalently bound spermine. The reactivity of anti-spermine antibodies was increased from 2- to 13-fold after dissociation and reassociation compared to that of non-dissociated area. The apparent reaction constant (Rapp.) of free IgG antibodies to spermine in the sera of 19 bronchopulmonary patients with cancer differed significantly from Rapp. values of IgG antibodies having this specificity in ten other patients with non-malignant disease.

Animals↗
PubMed1990

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies with a defined specificity. II. Application to immune complexes containing viral antigens in human sera.

The coupling of viral antigens from parainfluenza virus (PIV-1), cytomegalovirus (CMV) and human immunodeficiency virus (HIV-1) to chemically functionalized polystyrene plates has permitted us to develop a covalent enzyme-linked immunoassay (CELIA) for measuring the titers of free antibody (Ab) and immune complex (IC) bound Ab directed against each of these viruses. The method was first validated for experimentally produced IC (PIV-anti-PIV) and then applied to the analysis of IC in human sera. In the case of a renal transplant patient with CMV viremia whose free Ab titers were less than 100, the method unambiguously permitted the IC bound anti-CMV titers to be determined. In the case of a survey for HIV-1 Ab, it also allowed us to identify a sub-group of seropositives with IC anti-HIV. In view of the ease and rapidity with which CELIA can be performed, this technology should enable determinations of IC bound Ab of defined specificity to be undertaken routinely in seroepidemiological surveys.

Animals↗
PubMed1990

Synthesis of acetylenic spirobutenolide derivatives and evaluation of their growth inhibitory effect on cells in culture.

Acetylenic spirobutenolide amides and esters and their Mannich bases were synthesized to evaluate their growth inhibitory effect. The biological tests have used both normal and transformed cells and they have shown the selectivity of the prepared compounds. The ester derivatives presented the best selectivity comparable to that of daunorubicin.

Antineoplastic Agents↗
PubMed1989

The effect of a novel inhibitor of aldehyde dehydrogenase on viral replication.

The effect of AMPAL (4-amino 4-methyl 2-pentyne 1-al), an inhibitor of aldehyde dehydrogenase, on adenovirus type 5 (Ad5) replication was studied. AMPAL at 2 x 10(-4) M clearly reduced the cytopathic effect on HeLa cells but had no effect on cell growth at this concentration. Viral adsorption, penetration and protein synthesis were not affected by adding AMPAL at 2 hr post infection. When viral DNA synthesized in the presence of AMPAL was investigated, no significant inhibition was observed on either synthesis or the physicochemical properties of the neosynthesized DNA. However, there was a 4-fold increase in the amount of condensed DNA. In addition, AMPAL inhibited intracellular viral production (40%) and brought about concomitant inhibition of virus release (70%) into the medium. The absence of a quantitative relationship between the inhibition of viral DNA synthesis on one hand and that of viral production on the other may imply that the antiviral effect of AMPAL is indirectly mediated by the action of the malondialdehyde which has accumulated on some, as not yet identified, membrane constituent.

Adenoviruses, Human↗
PubMed1987

Protein-bound polyamines in the plasma of mice grafted with the Lewis lung carcinoma.

Protein-bound polyamines were isolated from the plasma of mice using antipolyamine antibodies covalently linked to magnetic latex spheres. Their subsequent separation by polyacrylamide gel electrophoresis (PAGE) showed that in plasma from normal mice, 3 proteins (27, 55 and 82 kDa) carrying polyamines could be visualized, whereas in mice bearing the Lewis lung carcinoma at least 8 other proteins of higher molecular mass (5 of 94, 110, 130, 145 and 160 kDa, and 3 of greater than 170 kDa) had bound polyamines. These protein-bound polyamines could be detected from the first week after tumour graft; they increased during the second and third week but decreased thereafter. These proteins were not bound by immunolatex spheres preincubated with spermine bound to a protein-carrier insulin. Moreover, the appearance of these protein-bound polyamines was not a consequence of the inflammatory process since in mice infected with heat-inactivated Brucella abortus, with the exception of a 65 kDa protein, polyamines were bound to the same proteins found in normal mice. In mice grafted with the Lewis lung carcinoma the concomitant decrease in transglutaminase-mediated polyamine (e.g. putrescine) binding capacity of plasma proteins provides additional evidence for the presence in vivo of polyamines already bound to plasma proteins.

Animals↗
PubMed1987

Malondialdehyde production from spermine by homogenates of normal and transformed cells.

The oxidation of spermine in vitro by a mixture of polyamine oxidase and diamine oxidase from pig kidney gives rise to malondialdehyde via 3-aminopropanol as the intermediate. Conversely, with spermidine, under similar experimental conditions, no evidence could be obtained for malondialdehyde formation within the limits of sensitivity of the assay (2.0 nmol). The activities of both these enzymes show about a 2-fold increase in normal rat kidney cells (LA31 NRK) transformed by the temperature sensitive mutant of Rous sarcoma virus (LA31) and incubated at the non permissive temperature (39 degrees C) compared to the activities in LA31 NRK at the permissive temperature (33 degrees C). These same enzymatic activities show no temperature dependent changes in normal rat kidney cells (NRK) or in these same cells infected by the wild type virus (NRK B77). In extracts derived from Friend erythroleukemic cells induced to differentiate by dimethyl sulfoxide or hexamethylene bis acetamide, spermine oxidation takes place more efficiently than in non induced cells. A rise in diamine oxidase activity is seen in LA31 NRK (39 degrees C) 12 h after the temperature shift, whereas morphological manifestations of normalcy are seen only at 48 h. The Km of diamine oxidase is 10(-6) M for putrescine and 10(-3) M for 3-aminopropanol. A possible mechanism involving the well documented acetylation of putrescine [23,26] is proposed for diverting intracellular putrescine away from cytosolic diamine oxidase and towards intramitochondrial monoamine oxidase.

Aldehydes↗
PubMed1987

[Demonstration of polyamines bound to plasma proteins during tumor growth].

Using antipolyamine antibodies covalently bound to magnetic latex spheres, we have been able to isolate from the plasma of mice bearing the Lewis lung carcinoma, 8 proteins with bound polyamines. These proteins are not found in the plasma of healthy mice or in those suffering with chronic inflammatory infection.

Animals↗
PubMed1986

Transglutaminase activity and putrescine-binding capacity in cloned cell lines with different metastatic potential.

An inverse correlation was found between cellular transglutaminase activity and metastatic potential of four cloned cell lines derived from a primary nickel-induced rat rhabdomyosarcoma. Cellular transglutaminase activity as assessed with endogenous cellular protein or exogenous methylated casein was greatest in the clone F9-4/8 which is the least metastasizing. When the putrescine-binding capacity of one cellular derived protein - fibronectin - was examined with exogenous transglutaminase, it was found that the fibronectin derived from the clone F9-4/8 showed the lowest binding capacity compared with those from the other clones. However, when the overall binding capacity of cellular proteins from each cell line was examined no differences could be detected. The results are discussed in the light of the well-known role of fibronectin in cellular adhesion.

Animals↗
PubMed1986

gamma-Glutamyltranspeptidase activity in human breast lesions: an unfavourable prognostic sign.

The activity of gamma-glutamyltranspeptidase (gamma GT) (EC 2.3.2.2) was examined by histoenzymatic labelling on frozen sections derived from normal breast tissue, benign lesions and carcinomas. In biopsies from normal tissue and benign lesions, labelling was very intense in lumina and in the apical pole of the cells lining the lumina whilst in the cytoplasm it was slightly positive. In 34 out of 70 carcinomas, gamma GT activity was either undetectable or slightly positive while in the remaining 36 there was intense activity. Statistical examination of the results revealed no obvious correlation of gamma GT activity with histological grade of the tumour, progesterone receptor content or classification of patients by pre- or postmenopausal status. A good correlation between gamma GT activity and the following unfavourable prognostic signs: lymph node metastases and absence of oestradiol receptors. Patients with gamma GT-negative tumours may have a more favourable prognosis than those with gamma GT-positive tumours.

Adenofibroma↗
PubMed1984

Red blood cell polyamines in mice bearing the Lewis lung carcinoma (3LL) and in patients with bronchopulmonary cancers.

Experimentally, during Lewis lung carcinoma (3LL) growth, the red blood cell (RBC) polyamine levels increase with tumor volume and are inversely correlated to tumoral concentrations of spermidine and spermine. The RBC level of spermidine is continually correlated to both the volume and the tumoral concentration of this polyamine. Clinically, high levels of RBC polyamine are observed in cases of squamous-cell carcinoma or anaplastic cancer, and not in cases of adenocarcinoma. RBC polyamine levels only permit the establishment of statistical differences between groups of patients. Therefore, clinical use of these molecules as tumor markers depends on an understanding of polyamine distribution within blood.

Adenocarcinoma↗
PubMed1984

A quantitative and qualitative study of the transglutaminase-mediated insertion of polyamines into plasma proteins from patients with bronchopulmonary cancer.

The transglutaminase-mediated incorporation of putrescine into proteins of plasma derived from 44 patients with histologically defined bronchopulmonary cancer was compared with that from an age-matched group of 18 patients hospitalized with non-malignant diseases. Two types of kinetic data were obtained over a range of putrescine concentrations 0.09 mM to 0.6 mM. Whereas the plasma from all the patients of the control group showed linear kinetics, that from only 24 patients of the cancer group showed linear kinetics. The remaining 20 showed kinetics typical of inhibition by excess substrate. PAGE analysis of [14C] putrescine-bound plasma proteins showed around 15 different bands. Among these putrescine-binding proteins, we identified, with the help of corresponding specific antisera, four protease inhibitors: alpha 2-macroglobulin, alpha 1-antitrypsin, alpha 1-antichymotrypsin and antithrombin III.

Acyltransferases↗
PubMed1984

The competitive inhibition of tissue transglutaminase by alpha-difluoromethylornithine.

The transglutaminase-mediated insertion of putrescine into casein was inhibited competitively by alpha-difluoromethylornithine (alpha-DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Preincubation of the amine acceptor (casein) or the enzyme itself with the inhibitor did not affect enzyme activity. Alpha-DFMO is a poorer substrate for transglutaminase (Km = 2.10 mM) than putrescine (Km = 0.17 mM). The inhibitory effect was also found with fibronectin as amine acceptor.

Acyltransferases↗