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M Brouwer

Publications and source records attributed to M Brouwer.

At least 91 records · Page 5Linked to original sources
PubMed1985

Characterization of three new variant type cell lines derived from small cell carcinoma of the lung.

Three new, well growing cell lines (GLC-1, GLC-2, and GLC-3) have been established from small cell lung carcinoma (SCLC) and characterized. A subclone (GLC-1-M13) markedly different from its parent line GLC-1 was also isolated and characterized. Cytogenetic analysis of the cell lines revealed deletions in the short arm of chromosome 3 as a most consistent chromosomal aberration. The deleted region was not identical in all metaphases, 3p(21-23) being the shortest region of overlap. Despite their SCLC origin GLC-1, GLC-2, and GLC-3 do not show pronounced SCLC differentiation features. Neurosecretory granula were very rare (GLC-1) or completely absent (GLC-2 and GLC-3), whereas the SCLC-related enzyme and hormone markers L-3,4-dihydroxyphenylalanine decarboxylase, neuron-specific enolase, creatine kinase BB, and bombesin-like immunoreactivity were variably expressed. Although the subclone GLC-1-M13 was derived from the poorly differentiated GLC-1, it behaved according to the above criteria as a differentiated "classic" SCLC cell line. When assessed with specific monoclonal antibodies the different cell lines appeared to express different subsets of intermediate filament proteins, indicative for different stages and directions of differentiation: "undifferentiated" (GLC-1 and GLC-2); "neural tissue related" (GLC-2); "simple epithelium" related (GLC-1-M13); and a combination of simple and squamous epithelium related (GLC-3). We conclude that GLC-1, GLC-2, and GLC-3 represent dedifferentiated forms of SCLC, related to the recently described "variant" type of SCLC, whereas the clonal derivate GLC-1-M13 behaves like a differentiated "classic" SCLC cell line.

Carcinoma, Small Cell↗
PubMed1984

Serum-dependent "cannibalism" and autodestruction in cultures of human small cell carcinoma of the lung.

In cell lines of human small cell carcinoma of the lung (SCCL) and in all subclones of one of the cell lines, cells were observed which completely interiorized other cells, leading to death of the interiorized cells and sometimes to complete autodestruction of the cultures. This phenomenon, which we have called "cannibalism," is also observed in fresh tumor biopsies from SCCL patients. Cannibalistic cells appeared to be of SCCL origin. "Cannibalism" is never observed in serum-free cultures but can be reinduced by serum exposure. It is likely that "cannibalism" may contribute to the frequent failure to establish SCCL cell lines in serum-containing medium. The potential to induce autodestruction of tumor cells in SCCL patients by as yet unknown serum factor(s) may be of therapeutic value.

Antigens, Surface↗
PubMed1983

Metal ion interactions with Limulus polyphemus and Callinectes sapidus hemocyanins: stoichiometry and structural and functional consequences of calcium(II), cadmium(II), zinc(II), and mercury(II) binding.

Hemocyanins are oligomeric metalloproteins containing binuclear copper centers that reversibly combine with oxygen molecules. The structural stability and functional properties of these proteins are modified by divalent cations. Equilibrium dialysis was used to study the reversible interaction of Callinectes sapidus and Limulus polyphemus hemocyanins with the divalent cations calcium, cadmium, zinc, copper, and mercury. The number of binding sites and association constants for each cation were obtained from an analysis of the binding data by a nonlinear least-squares minimization procedure. Spectral analysis showed Limulus hemocyanin to possess two mercury-reactive sulfhydryl groups per subunit (Kassoc = 2.02 X 10(45) M-1). Callinectes hemocyanin contains only one such group (Kassoc = 2.29 X 10(34) M-1). Cadmium and zinc are shown to substitute for calcium ions. Oxygen binding studies with Limulus hemocyanin showed that all five divalent metal ions increase its oxygen affinity. Calcium ions increase cooperativity of oxygen binding, while heavy-metal ions have an opposite effect. Binding of two mercuric ions per Limulus hemocyanin subunit irreversibly fixes the 48 subunit aggregate in a high-affinity noncooperative conformational state. These results offer a striking contrast to the functional consequences of heavy-metal ion interactions with Callinectes hemocyanin [Brouwer, M., Bonaventura, C., & Bonaventura, J. (1982) Biochemistry 21, 2529-2538]. The functional alterations associated with metal ion interactions are discussed within the context of an extension of the two-state model for allosteric transitions of Monod et al. [Monod, J., Wyman, J., & Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118]. Incubation of Limulus oxy- or deoxyhemocyanin with mercuric chloride results in the conversion of 60% of the binuclear copper sites to stable half-apo sites. The remaining active sites are stable with respect to mercury-induced copper displacement when oxygen is bridging both coppers. In the absence of oxygen these sites will eventually lose both copper atoms. Under the same conditions 50% of the binuclear copper sites of Callinectes deoxyhemocyanin are converted to half-apo sites. In this case oxygen completely protects against copper displacement [Brouwer, M., Bonaventura, C., & Bonaventura, J. (1982) Biochemistry 21, 2529-2538]. The binuclear copper center of Busycon carica is not affected at all, demonstrating profound differences between the active sites of hemocyanins of a chelicerate arthropod (Limulus), a crustacean arthropod (Callinectes), and a gastropod mollusc (Busycon).

Animals↗
PubMed1983

Cytotoxic effects of dexamethasone restricted to noncycling, early G1-phase cells of L1210 leukemia.

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10(-6)-10(-5) M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to 3H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.

Animals↗
PubMed1983

Isolation and characterization of subclones of L1210 murine leukemia with different sensitivities to various cytotoxic agents.

Two subclones of L1210 murine leukemia (L1210-46.1 and L1210-56.3) were isolated in the absence of selective agents. Subclone 56.3 appeared to be more sensitive than was subclone 46.1 to treatment with dexamethasone, 1-beta-D-arabinofuranosyl cytosine, vincristine, and X-irradiation. No differences between the parent cells and the two subclones could be observed in population-doubling time, cloning efficiency, number of chromosomes, and tumorigenic potential in DBA/2 mice. The subclones did not differ in the per cell number of glucocorticoid receptor sites. Animal experiments revealed an increase in life span of 65% in mice inoculated with cells from subclone 46.1 and of 130% of mice with subclone 56.3 after treatment with 1-beta-D-arabinofuranosylcytosine. The present results indicate that the L1210 wild-type murine leukemia cells contained stable subpopulations with a different but collateral sensitivity to various cytotoxic treatments. It is postulated that differences in drug sensitivity between cells are partly determined by cellular properties which are independent of the mechanism of action of any specific treatment.

Animals↗
PubMed1982

Heavy metal ion interactions with Callinectes sapidus hemocyanin: structural and functional changes induced by a variety of heavy metal ions.

Hemocyanins are oligomeric proteins that reversibly bind oxygen. The oxygen binding site is a binuclear copper center bound to the protein by amino acid side chains. The hemocyanin of the blue crab, Callinectes sapidus, occurs in vivo as a mixture of 25S dodecamers and 16S hexamers, whose oxygen binding properties are identical. Four heavy metals have been used as probes of structure and function in this hemocyanin system. Divalent cations of cadmium, copper, mercury, and zinc induced an indefinite self-association of the hemocyanin molecule. These higher ordered association states can be dissociated by ethylenediaminetetraacetic acid. Callinectes oxyhemocyanin possesses at least three mercury binding sites: (1) a sulfhydryl group which forms a mercaptide bond with a single mercuric ion, (2) a tryptophanyl side chain which forms a noncovalent 1:1 complex with mercuric ions with an association constant of 5.7 X 10(15) M-1, and (3) lower affinity site(s) involved in the self-association process also observed with cadmium, copper, and zinc. Sites 1 and 2 are most likely also involved in the binding of cadmium. Upon removal of oxygen from the active site of hemocyanin, an additional binding site becomes available for the reaction with mercury. Binding of mercury to this site leads to loss of one of the coppers from the binuclear oxygen binding site. Both the binuclear copper center and allosteric sites on the hemocyanin are affected by heavy metal binding. Cadmium and zinc ions increase the oxygen affinity; mercury and copper ions have the opposite effect. All four heavy metal ions decrease the degree of cooperative oxygen binding. The mercury-induced changes in oxygen binding by 25S Callinectes hemocyanin appear to be the result of that metal's interaction with the high-affinity tryptophan binding site. Mercury binding to the available sulfhydryl group in oxyhemocyanin occurs without functional consequences. Heavy metal, hydrogen, and chloride ions affect the affinity of the first or last oxygen molecules bound to the hemocyanin, which results in the appearance of multiple T (low oxygen affinity) and R (high oxygen affinity) states. Additionally, these ions shift the equilibrium between the low and high oxygen affinity states. The appearance of additional R states at high pH is accompanied by the cleavage of a tyrosine hydrogen bond.

Allosteric Site↗
PubMed1982

Phosphoenolpyruvate-dependent fructose phosphotransferase system of Rhodopseudomonas sphaeroides: purification and physicochemical and immunochemical characterization of a membrane-associated enzyme I.

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called "soluble factor" (SF) [Saier, M. H., Feucht, B. U., & Roseman, S. (1971) J. Biol. Chem. 246, 7819--7821]. SF has been partially purified by a combination of hydrophobic interaction and ion-exchange and gel-permeation chromatography. SF is similar to Escherichia coli enzyme I in its molecular characteristics and enzymatic properties. It has a molecular weight of 85 000 and readily dimerizes. Phosphoenolpyruvate and Mg2+ stabilize the dimer. The enzyme catalyzes the conversion of phosphoenolpyruvate into pyruvate and becomes phosphorylated in the process. The phosphoryl group is subsequently transferred to fructose in the presence of R. sphaeroides membranes. SF substitutes for E. coli enzyme I in fructose or glucose phosphorylation with E. coli enzyme II and HPr. The activities of SF with the R. sphaeroides PTS and the E. coli PTS reside on structurally distinct molecules as shown by their response to limited proteolytic digestion and by immunochemical studies. The activity of SF with the E. coli PTS arises during the isolation procedure and is most likely due to the removal of HPr-like protein from SF.

Chromatography↗
PubMed1980

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system. Evidence that the dimer is the active form of enzyme I.

In vitro kinetic measurements have been performed by using purified HPr, EI, and a membrane fraction of EII from the Escherichia coli phosphoenolypyruvate-dependent sugar transport system. These measurements reveal very large lag times in the formation of methyl alpha-glucoside phosphate which are a function of the EI and the EII concentrations. The lag times decrease with increasing concentrations of EI but they increase with increasing concentrations of EII. When EI, together with Mg2+ and phosphoenolpyruvate, is preincubated at 37 degrees C before starting the kinetic measurements, the lag time can be decreased or eliminated. We have shown that the process responsible for the lag time is the activation of EI by dimerization which is influenced by Mg2+ and phosphoenolpyruvate.

Biological Transport↗
PubMed1976

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: structural domains in the polypeptide chain.

alpha-Hemocyanin from the Roman snail Helix pomatia is composed of polypeptide chains with a molecular weight of 360000 +/- 30000. The cylindrically shaped hemocyanin molecule contains 20 of these large chains. The polypeptide chain has been split into components with molecular weights of: 210000, 154000, 147000, 112000, 120000, 98000, 55000, and 50000, by gentle proteolysis with enzymes of different specificities. Most of the fragments have molecular weights which are about 50000 or a multiple of 50000. Departure from these values, as found in the 112000 and 120000 fragments, is probably caused by the high carbohydrates content of these components. A mixture of these fragments has the same oxygen binding properties as the nondigested protein. Subtilisin converts the hemocyanin polypeptide chain, under appropriate conditions, almost completely into fragments of 50000 and 55000 daltons with conservation of the oxygen binding properties of the nondigested protein. We conclude from these studies that the polypeptide chain of Helix pomatia alpha-hemocyanin is folded into about seven compact tertiary structures, which are covalently interconnected. This chain of structural domains has been visualized. (Siezen and Van Bruggen (1974), J. Mol. Biol. 90, 77-89) by electron microscopy, which shows 1/20 hemocyanin molecules to be flexible structures consisting of 7-8 apparently spherical units of 55-60 A diameter.

Animals↗