Yehuda Ben-Shaul

Heavy riboflavin synthase from Bacillus subtilis: Quaternary structure and reaggregation☆

Heavy riboflavin synthase of Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 alpha-subunits (23.5 X 10(3) Mr) and 60 beta-subunits (16 X 10(3) Mr). The 10(6) Mr protein dissociates upon exposure to pH values above neutrality. Phosphate ions increase the stability at neutral pH. The dissociation induced by exposure of the enzyme to elevated pH is reversible in phosphate buffer at neutral pH. The stability of the enzyme at elevated pH values is greatly enhanced by the substrate analogue, 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. Electron micrographs of negatively stained enzyme specimens show spherical particles with a diameter of 15.6 nm. Various immunochemical methods show that the alpha-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-alpha-subunit serum, and riboflavin synthase activity is not inhibited by the serum. However, these tests become positive at pH values that lead to dissociation of the enzyme. Subsequent to dissociation of the native enzyme at elevated pH values, the beta-subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 S and 70 S. The average molecular weight was approximately 5.6 X 10(6). Homogeneous preparations have not been obtained. Electron micrographs show hollow, spherical vesicles with diameters of about 29 nm. The substrate analogue 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione can induce the reaggregation of isolated beta-subunits with formation of smaller molecules, which are structurally similar to native riboflavin synthase. A homogeneous preparation of reaggregated molecules was obtained by renaturation of beta-subunits from 6.4 M-urea in the presence of the ligand. The sedimentation velocity of this aggregate is about 7% smaller than that of the native enzyme. The molecular weight is 96 X 10(4). Electron micrographs show spherical particles with a diameter of about 17.4 nm. Inspection of the micrographs tentatively suggests the presence of a central cavity. It appears likely that these molecules, which are devoid of alpha-subunits, have the same number and spatial arrangement of beta-subunits as the native enzyme. All data are consistent with the hypothesis that the native enzyme consists of a central core of alpha-subunits surrounded by a capsid-like arrangement of beta-subunits. The number of beta-subunits and the shape of the protein suggest a capsid-like arrangement of beta-subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Junction formation in trypsinized cells of human adenocarcinoma cell line.

Abstract A human colon adenocarcinoma cell line was used to study junction formation and disassembly. Monolayered cells grown to confluency have desmosomes but no gap or tight junctions. Treatment with trypsin, while causing the breaking up of desmosomes to hemi-desmosomes, resulted in a rapid assembly of junctions. Tight junctions were formed in some recognizable steps: elevation of particle-free membrane ‘crests’, alignment of particles on these crests, and fusion of these particles to form typical tight junctions ridges. Gap junctions were also formed on particle-free membrane areas, but comparatively few such junctions were formed. Cycloheximide had no effect on the assembly of junctions. It is therefore assumed that pre-existing membrane particles were rearranged into junctions and that this rearrangement is probably due to the increased mobility of the trypsinized cell membranes. Transfer of trypsinized cells back into trypsin-free fresh medium resulted in internalization of phagocytic-like vesicles containing tight junctions elements.

A novel closed circular duplex DNA in bleached mutant and green strains of Euglena gracilis

Abstract Covalently closed circular duplex DNA, with a contour length of 3.13 ± 0.09 μm, has been isolated by cesium chloride-ethidium bromide equilibrium centrifugation from particulate fractions of permanently bleached mutant and green strains of Euglena gracilis Catenated dimers and oligomers (N = 2 to 6) were also observed. Circles were detected in log or stationary phase cells, dark-grown bleached or ethidium bromide-treated cells. Mitochondria either with or without deoxyribonuclease treatment released linear DNA by osmotic shock. Double labelling experiments showed that the circular DNA had a buoyant density of about 1.701 g·cm−3, which is different from that of nuclear, mitochondrial and chloroplast DNA.

The development and ultrastructure of lycopene bodies in chromoplasts ofLycopersicum esculentum

SummaryLycopene bodies are developed in tomato chromoplasts at temperatures permitting synthesis of lycopene. Their appearance seems to be in correlation with the formation of special rigid membranes. These membranes were not observed in chromoplasts of tomatoes ripened at 32‡ C, a temperature under which no lycopene is synthesized. Electron diffraction patterns of isolated lycopene bodies showed that the bulk of such a body is a lycopene crystal.Similarities between lycopene bodies of the tomato fruits and carotene bodies of carrot roots lead to the conclusion that classification of chromoplasts into distinct categories is valid only for certain stages of the chromoplast life cycle.

Structural modifications of the phycobiont in the lichen thallus

SummaryModifications in the fine structure of the algal component of two lichens,Aspicilia sp. andSquamarina crassa v.crassa, have been studied. It has been pointed out that fungal penetration is not essential for the mutual relationship between the two symbionts of the lichen thallus. The structural changes taking place during the life cycle of the phycobiont of the two lichens examined are not a response to fungal invasion.Careful examinations of serial sections revealed an interesting correlation between the growth pattern of the thallus and the distribution of the algal cells in the algal layer.

Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line.

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.

Actin-activated ATPase from human erythrocytes

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.

The Cytoskeletal Network Controls c-Jun Expression and Glucocorticoid Receptor Transcriptional Activity in an Antagonistic and Cell-Type-Specific Manner

ABSTRACT The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Müller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus.

Diabetic fibrillosis: A report of three cases

Three patients with diabetes are described. Two patients had a mild, well controlled, hyperglycemic syndrome; five years after its detection signs of nephropathy and retinopathy appeared. Both died of renal failure ten years after detection of diabetes. The third patient died at the age of forty-seven of recurrent myocardial infarction. During her terminal hospitalization uremia and hyperglycemia were first discovered. Histologic findings in postmortem material were identical in all three cases. Heavy deposits of pediodic acid-Schiff-positive, colloidal iron and Congo red negative material were found in the blood vessels of virtually all organs examined. The blood vessels involved ranged from capillaries, venules, arterioles to arteries of large size. In some areas the material was seen also in connective tissue outside of blood vessels. Electronmicroscopic examination revealed that the PAS-positive material was composed of fibrils, approximately 100A wide. These findings indicate the presence of a systemic disease of connective tissue in our cases, resembling systemic amyloidosis in extent, location and nature. We suggest the name, diabetic fibrillosis.

The effect of modifying the culture medium on cell polarity in a human colon carcinoma cell line

The human colonic cancer cell line HT29, when grown in DMEM, forms a morphologically unpolarized cell culture in which the cells are covered with irregular microvilli and devoid of belt zonula occludens type tight junctions. However, by modifying the culture medium and growing the cells in RPMI, a different morphology was obtained. A large number of intracellular luminae appeared and at late confluency 90-95% of cells exhibited an apical brush border after four subsequent passages. Junctional complexes and a well developed zonula occludens were revealed under the apical brush border. Immuno-electron microscopical localization of specific markers, sucrase isomaltase (SI), secretory components (SC) and beta 2 microglobulin (beta 2M) showed that SI was limited to the apical surface, whereas 2M and SC were located at the basolateral surfaces. These results indicate that modification of culture conditions affects the ability of HT29 cells to express epithelial cell polarity.