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S W Tanenbaum

Publications and source records attributed to S W Tanenbaum.

At least 37 records · Page 2Linked to original sources
PubMed1979

Convenient procedures for the biosynthesis, isolation, and isotope labeling of cytochalasins.

Efforts to improve small-scale yields of useful cytochalasins by fermentation resulted in selection of an enriched aflatoxin medium which increased yields by fivefold over those reported in the literature. With Helminthosporium dematoideum and Zygosporium masonii in stationary culture for 3 weeks, cytochalasins B and D were obtained in quantities approaching 700 and 500 mg/liter, respectively. It appears that the critical component in this growth medium is factors associated with whole wheat. By using these procedures, coupled with improvements in isolation, supplementation with two radioactive phenylalanine species readily produced [14C]- or [3H]cytochalasin B. Oxidation of carrier-free radioactive cytochalasin B to cytochasasin A readily provided this labeled congener as well. The isotopic ocnversion of precursor to crystalline products that met analytical criteria ranged from 2 to 4% of the administered radioactivity.

Culture Media↗
PubMed1979

Physiological responses of bacteria to cytochalasin A: effects on growth, transport, and enzyme induction.

Cytochalasin A at 5 to 25 microgram/ml (1.0 x 10(-5) to 5.2 x 10(-5) M) inhibited the growth of three gram-positive bacteria, Arthrobacter sialophilus, Staphyloccus aureus, and Bacillus amyloliquifaciens, but had little or no effect on the growth of three gram-negative bacteria, Excherichia coli, Pseudomonas maltophilia, and Aeromonas proteolytica. A. sialophilus and S. aureus recovered spontaneously from cytochalasin A-mediated growth inhibition after a considerable lag period, which was dependent on the drug dose. It was demonstrated that this long-term recovery did not involve selection of resistant variants. Cytochalasin A had no detrimental effect on cell viability in A. sialophilus or S. aureus, but caused lysis of B. amyloliquifaciens. The drug prevented enzyme inductions and inhibited transport of valine, uridine, and glucose in the gram-positive organisms. It had little or no effect on these processes in the gram-negative organisms. In studies with A. sialophilus, the drug inhbitied respiration of exogenous substrates, but did not depress endogenous respiration. These results constitute the first unequivocal evidence for the bacteriostatic properties of this class of compounds and indicate that cytochalasin A halts various physiological processes in gram-positive bacteria primarily by inhibiting solute transport.

Bacteria↗
PubMed1978

Purification and properties of Arthrobacter neuraminidase.

Neuraminidase (EC 3.2.1.18) from an Arthrobacter species was purified homogeneity by conventional procedures (yield approx. 1 mg/1) and was judged to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Gel electrofocusing of neuraminidase revealed 1 major band (85-90%), pI 5.35 +/- 0.05, and 6 minor bands, whose pI ranged from 5.25 to 5.70, and each of which had catalytic activity. Arthrobacter neuraminidase is a monomeric glycoprotein of molecular weight 88 000, has an apparent Km of 7.8-10(-4) M for N-acetylneuraminlactose, is insensitive to inhibition by N-acetylneuraminic acid, and is about 2% carbohydrate by weight. The amino acid composition as well as the galactosamine and glucosamine content was determined. The enzyme can hydrolyze (alpha, 2-3), (alpha, 2-6), (alpha, 2-8) linkages. The active size of the enzyme appears to be inaccessible since no inhibition was observed by reagents known to modify sulfhydryl, lysyl, carboxyl, histidinyl, and argininyl residues. In contrast, N-bromosuccinimide at a 60-fold molar ratio to enzyme, gave complete inhibition. These results suggest that a tryptophan residue is essential for catalysis.

Amino Acids↗
PubMed1978

Induction and regulation of neuraminidase synthesis in Arthrobacter sialophilus.

A variety of N-acetylneuraminic acid (AcNeu) derivatives and analogs were examined as inducers of the extracellular neuraminidase of Arthrobacter sialophilus. Neuraminidase inductions were primarily studied with tryptone-yeast extract-grown cells after washing and resuspension in a defined replacement medium. The addition of readily metabolizable carbon sources to the latter, such as 0.1% casein hydrolysate, glutamate, or glucose, enhanced enzyme synthesis. Enzyme appearance occurred after a lag in the uptake of inducers, suggesting the participation of a co-inducible transport system. Neuraminidase formation during exponential growth in the presence of AcNeu ceased after depletion of this end product from the medium. It was found, besides AcNeu, that its methyl ester, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid methyl ester are each active inducers, whereas beta-anomers of AcNeu-ketosides are not. These results, in comparison to known enzyme specificity, have revealed significant differences and parallels between the inductive and catalytic processes for neuraminidase. In particular, it would appear that the free carboxylate and oxygenation at C-2 of AcNeu, essential for enzyme catalysis with traditional AcNeu substrates, are not necessary for induction and, furthermore, that transition state analogs can specifically induce this enzyme. The failure to observe catabolite repression in this system is discussed in relation to the intermediary metabolism of the genus Arthrobacter.

Arthrobacter↗
PubMed1978

Effects of cytochalasin A on the morphology of plasmodia and sclerotia of Physarum polycephalum.

Cytochalasin A (CA) at 1.6 X 10(-5)M and lower concentrations produced disruptive effects upon plasmodia, sclerotia, and spherule forms of Physarum polycephalum. CA effects upon either micro- or macroplasmodia included: cytoplasmic condensation, plasmodial contraction, and scission at the plasma membrane. The latter manifestation was most dramatically observed by scanning electron microscopy. Electron microscopy of drug-treated palsmodia confirmed the above phase contrast light-microscopic results and revealed, in addition, vacuolar enlargement, decreased membrane invaginations, and the presence of condensed particles within the plasmodium and at the plasma membrane. These results of drug action were not reversed by washing of exposed plasmodia. Germination of the slcerotial and spherule forms of Physarum was CA-delayed by 12 and 96 hours, respectively. Approximately 10% of drug-treated sclerotia were found to have been burst apart. These dramatic CA effects were nullified by preincubation of the drug either with L-cysteine or with beta-mercaptoethanol; however, iodoacetamide (10(-6)M) gave no such morphologic consequences. Cytochalasins B or D at comparable concentrations were without activity. It is concluded therefore that CA effects upon the myoxomycete reflect specific acceptor responses.

Cysteine↗
PubMed1977

The binding sites of cytochalasin D. I. Evidence that they may be peripheral membrane proteins.

Binding sites for tritiated cytochalasin D (3H-CD) on the isolated plasma membrane from HEp-2 cells were reversibly inactivated, but not dissociated from the membrane, by dialysis in 0.6 M KCl. Activity was restored by subsequent dialysis in 0.06 M KCl. Treatment with 0.2 mM ATP at low ionic strength also inactivated these sites, apparently irreversibly. Extraction of the membrane with 6% Triton X-100 removed 75% of its protein, resulting in a two-fold increase in specific binding activity for 3H-CD. Both high and low affinity binding sites were retained by the detergent-extracted membrane; at least 60% of the high affinity sites were resistant to this treatment. Evidence is presented for the attachment to the HEp-2 plasma membrane of both actin and myosin. The results support the tentative conclusion that plasma membrane binding sites for 3H-CD are peripheral proteins on the cytoplasmic face of the membrane. They are consistent with the hypothesis that myosin may be the location of the high affinity binding site and actomyosin may be the low affinity site. Comparison of these observations with those reported for the congeneric drug, cytochalasin B, suggests that CD binding sites differ from the high affinity site for cytochalasin B.

Actomyosin↗
PubMed1977

The binding sites of cytochalasin D. II. Their relationship to hexose transport and to cytochalasin B.

Cytochalasin B (CB) was able to compete with tritiated cytochalasin D (3H-CD) for binding sites in HEp-2 cells. The pattern of inhibition suggested that CB associates with a low affinity class of CD binding sites. Glucose and maltose did not inhibit binding of 3H-CD to isolated HEp-2 plasma membrane. Inhibition of hexose transport by CD was negligible, but CD did not block the potent inhibition of this transport by CB. These results indicate that CD does not bind to the high affinity CB receptor reportedly associated with the hexose transport system, and that this receptor cannot mediate the morphological effects of CD. Both CD and CB induced contraction-zeoisis in HEp-2 cells; CB was less potent than CD, and their effects appeared to be additive. It was concluded that the high affinity binding sites for CD and CB are different, but that these congeners share a low affinity site. Both high and low affinity sites for CD appear to mediate its morphological effects; only the low affinity class appears to be involved for CB. Possible identification of the common low affinity binding site as actomyosin (detailed in Tannenbaum et al., '77) is further discussed.

Binding Sites↗
PubMed1977

Properties of an inducible extracellular neuraminidase from an Arthrobacter isolate.

The elective isolation of a soil microorganism, tentatively assigned to the genus Arthrobacter, which produced an extracellular neuraminidase is described. The secretion of neuraminidase from washed cells in minimal medium required the presence of sialo-containing glycoproteins, whereas free N-acetyl-neuraminic asid of N-acetylmannosamine were poor inducers. No enzyme could be dected in the induction fitrated of cells, in the absence of inducer or in the culture filtrate of cells grown in a complete medium. The routine enzyme inducer was a hot-water extract of "edible bird's nest." Mild acid treatment (0.05 N H2SO4) of this extract increased enzyme activity two--to threefold and the specific activity about eightfold. Neuraminidase induction with acid-treated bird's nest was manifested at a linear rate for 6 h without increase in cell number. No other anticipated glycohydrolase or protease activities were foud. The amount of enzyme located within the cells was barely detectable as compared to that found in the induction filtrate. Experiments with chloramphenicol or chlortetracycline indicate that de novo protein synthesis was required for neuraminidase production and that this exoenzyme was not released from a preformed pool. Neuraminidase from this source has an apparent molecular weight of 87,000, a pH optimum of 5 to 6, and an apparent Km of 2.08 mg/ml for collocalia mucoid and 3.3 X 10(-3) M for N-acetylneuraminlactose and is insensitive both to Ca2+ ions and ethylenediaminetetraacetic acid. Preliminary studies indicate that the enzyme can hydrolyze alpha-2,3-, alpha-2,6-, or alph-2-8-N-acetylneuraminylglycosidic linkages. From total activity data and purification criteria, it would appear that this isolate can produce about 5 mg of enzyme per liter of induction medium.

Arthrobacter↗
PubMed1977

Arthrobacter sialophilus sp. nov.; a neuraminidase-producing coryneform.

A new species of Arthrobacter is described for which the name A. sialophilus is proposed. The organism was obtained by elective culture from soil using Collocalia mucoid, and can readily be induced in replacement minimal media with appropriate substrates, to form the enzyme neuraminidase. It undergoes sphere-rod-sphere morphogenesis, is gram-variable, non-motile, aerobic, catalase-positive, oxidase-negative, fails to release acid or gas from saccharides, has a GC content of 56.0 mol % and can grow at temperatures up to 45 degrees C. The peptidoglycan composition of its spherical form contains neither diaminopimelic acid nor arabinose as components, but does manifest the presence of glycine. Its generation time at 30 degrees C on tryptone-yeast extract medium is 1.6 h. Accessory growth factors do not appear to be required. The organism, though colorless in the dark, provides a bright yellow water-insoluble pigment after exposure to light. The foregoing combination of properties distinguishes this isolate from other well-described Arthrobacter spp.

Arthrobacter↗
PubMed1975

Subcellular localization of binding sites for cytochalasin D: evidence from activation energies.

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at greater than 11.5 degrees C, 18 kcal/mol at less than 11.5 degrees C) was taken as evidence that [3H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [3H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).

Binding Sites↗
PubMed1975

Action of cytochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface.

The projection of knobby protuberances at the cell surface (zeiosis) is a general cellular response to cytochalasin D (CD), resulting from herniation of endoplasm through undefended places of the cortex during cell contractions and displacement of microfilaments induced by CD. Zeiosis is prevented by agents that interfere with the contractile response to CD, such as inhibitors of energy metabolism or cyclic AMP. The developed protrusions, which remain relatively stable in the presence of CD, contain chiefly mono- or subribosomes, and occasionally other organelles normally resident in endoplasm; compact microfilament felt occupies their bases and extends into their proximal stalks. Protein synthesis in the knobs is less than half of that in the polyribosome-containing endoplasm residual in the main body of the cell. Knobs first protrude singly near the margin of the contracting cells and rapidly cluster into small groups in the periphery even at lower temperature. The clusters then migrate centripetally and coalesce into a large aggregate near the apex of the immobilized and retracted cell: this movement is energy- and temperature-dependent. Aggregation is more prominent and stable in cell lines of epithelial derivation than in fibroblastic or other lines in which nuclear extrusion occurs more readily. The latter is regarded as a special manifestation of zeiosis. Macromarkers, such as latex spherules, migrate like the zeiotic knobs on the cell surfaces in the presence of CD. The aggregated knobs, although persistent for days in the presence of CD, are rapidly recessed after withdrawal of the agent as ruffling is resumed and the cells spread. These movements are discussed in terms of current concepts of mobility of the cell membrane.

Amino Acids↗
PubMed1974

Action of cytochalasin D on cells of established lines. I. Early events.

HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2-0.5 microg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G(1) are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G(2). CD inhibits transport of [(14)C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.

Animals↗
PubMed1974

Action of cytochalasin D on cells of established lines. II. Cortex and microfilaments.

Cells in culture exposed to cytochalasin D (CD) rapidly undergo a long-sustained tonic contraction. Coincident with this contracture the thin microfilaments of the cortex become compacted into feltlike masses. The ravelled filaments of these masses remain actinlike and bind heavy meromyosin; they are not disrupted or disaggregated, but rather, appear to represent a contracted state of the microfilament apparatus of the cell cortex. On continued exposure to CD, 'myoid' bundles, containing thick, dense filaments, and larger fusiform or ribbonlike, putatively myosinoid, aggregates may appear. These appearances are interpreted as consequences of a state of hypercontraction without relaxation induced by CD. They do not occur in CD-treated cells prevented from contracting by inhibitors of energy metabolism, and are readily reversible on withdrawal of CD. Extensive ordered arrays of thin microfilaments develop in cells which are reextending during early recovery.

Adenosine Triphosphate↗