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PubMed1979

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.

Biological Transport↗
PubMed1979

The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.

Cations, Monovalent↗
PubMed1985

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica.

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.

Animals↗
PubMed1979

Polymorphonuclear leukocyte-inhibitory factor of Bordetella pertussis. II. Localization in the outer membrane.

The outer and inner membranes and cytoplasm of spheroplasts of a strain of phase I B. pertussis were fractionated by density gradient centrifugation. The high density vesicles of the outer membranes isolated had the "Pili" characteristic of the bacteria and the same antigenicty as the bacterial surface. Activities for inhibition of polymorphonuclear leukocytes were also almost exclusively localized in this outer membrane fraction. The histamine-sensitizing activity was more dispersed, but its specific activity was also highest in the outer membrane fraction. These results suggest that molecules carrying these activities, which are probably different entities together with the tissue-adhesive pili, form a virulence complex on the surface of phase I organisms of B. pertussis.

Antigens, Bacterial↗
PubMed1980

[Structural organization of the bacterial nucleoid using endonucleolysis].

The fragmentation of bacterial deoxyribonucleoprotein (bDNP) in the spheroplasts of Escherichia coli, Serratia marcescens, Pseudomonas fluorescens and Micrococcus luteus by bacterial intracellular Ca2+ or Ca2+, Mg2+-dependent endonucleases in situ was studied. An electrophoresis of the extracted nuclease-split bDNP revealed the presence of high molecular weight (nuclease-resistant) and low molecular weight multiple fragments (100--120 nucleotide pairs). The electrophoretic mobility of the smallest nuclease-split DNA fragments in all bacterial species under study was similar, indicating the orderly structure of bDNP. Two total fractions whose electrophoretic mobility corresponded to that of the histones H2a and H2b from calf thymus were prevalent in the spectrum of acid-soluble bDNP proteins of gram-negative species. The heterogeneity of DNP with respect to its sensitivity to nucleases, is interaction with membranes and protein distribution pattern were revealed by treatment of the bacterial nucleoid with endogenous endonucleases, which probably reflects differences in the functional state of individual sites of the genome.

Animals↗
PubMed1984

[Electron microscopic characteristics of the action of penicillin and vancomycin on Rickettsia conorii and Rickettsia akari in vitro].

The possibility of L-transformation for two species of Rickettsia of the tick group (R. conorii and R. akari) was studied with light and electron microscopy. The experiments were performed on the cultures of L-cells (murine fibroblasts). Penicillin and vancomycin were used as the transforming agents. Electron microscopy revealed clear changes in the morphological structure of both species when the doses of penicillin and vancomycin were 50-100 and 250-1500 micrograms/ml, respectively. The antibiotics had a morphologically analogous effect on both species of Rickettsia. The resulting morphological structures were large oval spheroplast-like cells with significant expansion of the periplasmic space up to 1 X 1.75 micron under the effect of penicillin and 0.5 X 0.7 micron under the effect of vancomycin. R. akari was somewhat more sensitive to the antibiotics. Electron microscopy of the morphological changes in the Rickettsia induced by penicillin and vancomycin showed that they were similar to the changes observed in gram-negative bacteria on their L-transformation and to the penicillin forms of chlamydia (galprovia) and R. prowazekii. They may be considered as a phase of unbalanced growth and represent the initial stage of L-transformation.

Cell Wall↗
PubMedDate not supplied

[Comparative study of the oxygen reduction in photosystem II of the cyanobacteria, Anacystis nidulans and Anabaena variabilis].

Oxygen reduction in the photosystem II (PS II) of thylakoid membranes from the cyanobacteria Anacystis nidulans and Anabaena variabilis was studied in the system SiMo + DCMU in whose presence competitive electron transport to SiMo and O2 in possible. The reagents of the Mehler reaction were used; these reagents activate oxygen uptake by interacting with the reduced forms of oxygen (malonate and oxalate with O2; glyoxylate and catalase + ethanol with H2O2). The use of the reagents as shown that there was a considerable electron transport to O2 which decreased the rate of SiMo reduction and visible oxygen evolution. Oxygen was reduced to O2 and H2O2 in the photosystem II of A. nidulans whereas practically no generation of H2O2 was observed in the photosystem II of A. variabilis. Malate was found to be capable of activating oxygen uptake in the PS II of the cyanobacteria by interacting with O2. Oxalate was capable of this interaction only in the membranes of A. variabilis, but not in the membranes of A. nidulans where it competed with water as an electron donor. A decrease in SiMo reduction and oxygen evolution in A. nidulans in the presence of glycolate was due to oxidation of glyoxylate which was formed in the course of enzyme-catalyzed oxidation of glycolate by the membranes possessing the activity of glycolate oxidase, and the subsequent interaction of glyoxylate with H2O2. SiMo reduction by the cyanobacterial membranes and by the spheroplasts of A. variabilis, in contrast to the chloroplasts of higher plants, did not decrease in the presence of DCMU, but went on at a linear rate until the substrate was completely exhausted. The role of reduced O2 forms in metabolism of a phototrophic cell is discussed.

Cell Membrane↗
PubMed1982

[Permeability of the membrane of the Escherichia coli envelope for ethidium bromide].

The interaction of ethidium bromide, a fluorescent dye, with Escherichia coli cells was studied. The envelope of intact cells was shown to be impermeable for ethidium bromide molecules. The dye penetrated however into E. coli spheroplasts. The barrier properties of the cell envelope against ethidium bromide were ruptured if the cells were treated with EDTA. The results suggest that the outer membrane serves as a principal barrier against penetration of ethidium bromide inside the cells while the cytoplasmic membrane of E. coli is permeable for the dye.

Cell Membrane↗
PubMed1980

Intracellular localization of two molecular forms of membrane acid protease in Aspergillus oryzae.

The intracellular localization of two forms of membrane-bound acid protease (M1 and M2) [EC 3.4.23.6] of Aspergillus oryzae (Tsujita, Y. & Endo, A. (1978) Eur. J. Biochem. 84, 347-353) was investigated. When the mycelia were treated with wall-lytic enzymes, M2 remained in the cells but most of M2 was solubilized and released. The cell wall fraction obtained by mechanical disruption of the mycelia contained less than 5% of the total acid protease activity in the cells. Subcellular fractionation of the membranes obtained from burst spheroplasts showed that the acid protease was present in both rough and smooth microsomes. Acid protease M1 was predominant in the former and M2 in the latter, possibly on the surface of the cytoplasmic membranes.

Aspergillus↗
PubMed1982

Active transport of maltose in Escherichia coli K12. Role of the periplasmic maltose-binding protein and evidence for a substrate recognition site in the cytoplasmic membrane.

The active transport of maltose in Escherichia coli requires the products of five genes. These include a water-soluble periplasmic maltose-binding protein, three cytoplasmic membrane proteins, and an outer membrane protein. In order to evaluate the role of the maltose-binding protein in active transport, a nonpolar internal deletion of the structural gene for the maltose-binding protein was constructed. A strain which contains this deletion is unable to grow on maltose at an external concentration of 25 mM, even when the remaining components of the transport system are synthesized constitutively. This demonstrates that the periplasmic maltose-binding protein is essential for detectable translocation of maltose across the cytoplasmic membrane. Mal+ revertants of the deletion strain were obtained. In one of these strains, the remaining components of the maltose transport system gained the ability to translocate maltose across the membrane independently of the periplasmic binding protein. Maltose transport in this revertant strain is specific for maltose; it is not inhibited by other alpha and beta glucosides and galactosides. In contrast to the wild type, transport activity in the Mal+ revertant strain is retained by spheroplasts. The cytoplasmic membrane components of the maltose transport system in the revertant appear to form a substrate recognition site. It is likely that this site exists in wild type cells but is available only to substrate molecules that are bound to the maltose-binding protein. A model for the operation of the transport system is presented. In this model, the substrate recognition site in the cytoplasmic membrane is exposed to alternate sides of the membrane.

ATP-Binding Cassette Transporters↗
PubMed1981

Etiology of granulomatous disease of the gut (Crohn's disease).

This report is based on a review of the various investigations attempting to determine the etiology of Crohn's disease. It has paid special attention to three main possible etiologic mechanisms, reported in the literature up to the time of this review; namely, immunologic, unknown transmissible agents or known such as L-forms, protoplasts, or spheroplasts, viral or infectious RNA and/or DNA, and a combination of the preceding causative possibilities. It has discussed the unrewarding immunological mechanisms, the failure to confirm the various investigations suggesting a transmissible agent and/or the speculations concerning the possible combination of factors. It also has included the remarks of Sacchar and Auslander, who stated that four important pieces were missing to allow acceptance of transmissible agent as the cause of Crohn's disease. This review has demonstrated that up to the present time the etiology of Crohn's disease has remained a puzzle and concluded that further investigations are necessary to resolve the etiology of Crohn's disease with the suggestion that evidence for an infectious agent will require experiments that satisfy Koch's postulates.

Animals↗
PubMed1995

High membrane fluidity is related to NaCl stress in Candida membranefaciens.

The effect of hypersaline stress on the lipid composition of the salt-tolerant yeast Candida membranefaciens was studied. Fatty acid analyses of the plasma membrane showed a growth phase- and dose-dependent increase in the level of linolenic acid (C18:3) in 1.35 M NaCl-stressed cells. Palmitoleic acid (C16:1) was completely undetectable at all phases of the life cycle. Changes in the levels of other fatty acids were insignificant. The degree of unsaturation of fatty acids in the plasma membranes was higher in presence of 1.35 M NaCl. The fluorescence polarisation value of DPH (1,6-diphenyl- 1,3,5-hexatriene) in the spheroplasts of the stressed cells was lower as compared to the control cells, indicating the fact that a higher membrane fluidity favours osmotic adaptation against NaCl stress. Among different phospholipids, levels of Phosphatidylinositol and Phosphatidylethanolamine were elevated in the salt-adapted cells as compared to their controls. The levels of Phosphatidylcholine and cardiolipin did not change significantly in response to hypersaline stress. The study points out that hypersalinity signals affect the lipid composition which in turn affects the membrane fluidity of C. membranefaciens.

Candida↗
PubMed1976

Methylated, blocked 5' termini of yeast mRNA.

mRNAs of the yeast, Saccharomyces cerevisiae, have modified 5' termini similar but not identical with those present in the higher eukaryotes. Poly(A)-containing RNA isolated from spheroplasts or from polysomes of cells labeled with [methyl-3H]methoionine or 32Pi contain methylated, blocked 5'-terminal structures of composition: m7G(5')pppAp and m7G(5')pppGp, occurring in a relative distribution of 75% and 25%, respectively. Furthermore there is no N6-methyladenosine nor any other methylated nucleoside in the entire molecule of poly(A)-containing RNA. Experiments with a mutant, temperature-sensitive for the transport of mRNA to cytoplasm, suggest that the methylation and addition of the blocking group take place in the nucleus and that the genetic lesion in the mutant does not affect these mechanisms.

Methionine↗
PubMed1976

[A study of Proteus L forms during reversion by means of electron microscopy and ultrathin sections].

The method of scanning electron microscopy was applied to the study of the Proteus L-forms which reversed directly on the millipore filters. It appeared that reversion was accompanied by elongation of the cell, piriform cells forming as the result. Filamentous cells segmented into short fragments and large bodies with a spongy surface structure with numerous ruptures were also revealed in the reversing culture. The method of ultrathin sections showed the other membrane in many cells to be fragile and readily twisting forming vesicles collecting into chains. As supposed, this is connected with degenerative changes involving the outer membrane of the paternal L-forms at the early reversion periods. An interrupted structure of the outer membrane was also demonstrated in many cells. It is supposed that in this case a synthesis of the outer membrane de novo at the later stages of reversion is dealt with. Phenomenon of "extrusion" of the multilayer structures into the periplasmic space, characteristic of spheroplasts, is described. A marked similarity of the morphological picture of reversion and at the initial stages of L-transformation, i.e. of the processes representing a looking glass reflection of one another, is described.

Cell Membrane↗
PubMed1993

Preparation of a monoclonal antibody specific for a 48-kDa protein from mitochondrial nucleoids of the yeast, Saccharomyces cerevisiae.

Monoclonal antibodies (mAbs) were raised against yeast mitochondrial nucleoids (mt-nucleoids). In an analysis by a combination of immunofluorescence microscopy and staining with 4',6-diamidino-2-phenylindole (DAPI), one of them, designated YMN-1, distinctly stained mt-nucleoids, which were visible as dots, in spheroplasts and in isolated mitochondria. However, staining of isolated mt-nucleoids was rather weak. YMN-1 mAb recognized a 48-kDa protein in immunoblots of both mitochondrial and mt-nucleoid proteins. The 48-kDa protein was a minor component of mt-nucleoid proteins and was separated from extract of both mitochondria and mt-nucleoids by immunoaffinity chromatography. The affinity-purified 48-kDa protein reassociated with mt-nucleoids when mixed with isolated mt-nucleoids, as monitored by immunofluorescence microscopy. The results suggest that a large amount of 48-kDa protein is associated with mt-nucleoids in vivo, and that lysis of mitochondria by the treatment with detergent releases a considerable amount of this protein from mt-nucleoids during the isolation of mt-nucleoids.

Animals↗
PubMedDate not supplied

[Yersinia L-transformation in experimental pseudotuberculosis].

An experiment on guinea pigs infected with virulent Y. pseudotuberculosis has revealed for the first time that in the host body Yersinia cells undergo ultrastructural changes, characteristic of L-transformation. These changes are manifested by damages in the cell wall and peptidoglycan layer of bacteria, dramatic expansion of the peri-plasmatic space, appearance of spheroplast bacterial cells. Myelin-like structures are formed around dead bacteria. These changes reveal one of the possible mechanisms which may render Yersinia harmless in convalescent patients, as well as contribute to the development of relapses of Y. pseudotuberculosis infection.

Animals↗