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PubMed1995

Ultrastructural studies of the myenteric plexus and smooth muscle in organotypic cultures of the guinea-pig small intestine.

External muscle and myenteric plexus from the small intestine of adult guinea-pigs were maintained in vitro for 3 or 6 days. Myenteric neurons and smooth muscle cells from such organotypic cultures were examined at the electron-microscopic level. An intact basal lamina was found around the myenteric ganglia and internodal strands. Neuronal membranes, nuclei and subcellular organelles appeared to be well preserved in cultured tissues and ribosomes were abundant. Dogiel type-II neurons were distinguishable by their elongated electron-dense mitochondria, numerous lysosomes and high densities of ribosomes. Vesiculated nerve profiles contained combinations of differently shaped vesicles. Synaptic membrane specializations were found between vesiculated nerve profiles and nerve processes and cell bodies. The majority of nerve fibres were well preserved in the myenteric ganglia, in internodal strands and in bundles running between circular muscle cells. No detectable changes were found in the ultrastructure of the somata and processes of glial cells. Longitudinal and circular muscle cells from cultured tissue had clearly defined membranes with some close associations with neighbouring muscle cells. Caveolae occurred in rows that ran parallel to the long axis of the muscle cells. These results indicate that the ultrastructural features of enteric neurons and smooth muscle of the guinea-pig small intestine are well preserved in organotypic culture.

Animals↗
PubMed1991

Effects of Ca2+ and ionophore A23187 on protein synthesis in intact rabbit reticulocytes.

1. Amino acid incorporation in intact rabbit reticulocytes was unaffected by depletion of Ca2+ with EGTA. 2. The Ca2+ ionophore A23187 strongly inhibited incorporation in reticulocytes incubated in 1 mM Ca2+ but not in EGTA. Polysomal profiles and average ribosomal transit times of cells treated with Ca2+ ionophore at 1 mM Ca2+ were characteristic of translational elongation block. 3. The behavior of reticulocytes with respect to Ca2+ and A23187 contrasts with that of nucleated cells possessing endoplasmic reticulum in which protein synthesis is inhibited at translational initiation by either Ca2+ depletion or by exposure to Ca2+ ionophore.

Animals↗
PubMed2005

Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources.

The aerobic degradation of phenol, chlorobenzene and dichlorobenzene as a sole carbon source has been observed in bacterial Gram-positive strain G2PT isolated from a wastewater bioprocessor. Cells display branching mycelia fragmenting into rod and coccoid elements when grown on TSA. Aerial hyphae formation occurs when grown on phenol and chlorinated aromatics as the sole carbon source. Growth was observed at up to 0.75% phenol as a sole carbon source, indicating a strong tolerance for the compound. The 16S rRNA gene sequence shares the greatest similarity with members of the Rhodococcus genus, with the closest shared nucleotide identity of 98% with the aromatic toxin degrading bacteria Rhodococcus zopfii DSM 44108T. Neighbor-joining and parsimony analysis of Corynebacterineae 16S rRNA gene sequences consistently places strain G2PT in a clade shared with R. zopfii within the Rhodococcus rhodochrous subclade. Based on a unique polyphasic profile involving phenotypic, ribosomal DNA sequence analysis, DNA-DNA hybridization, mol% DNA G+C content and fatty acid composition, G2PT is proposed to represent a previously uncharacterized, novel species in the genus Rhodococcus. The name Rhodococcus phenolicus is proposed for the isolate with the type strain G2PT (= DSM 44812) (= NRRL B-24323) [corrected]

Bioreactors↗
PubMed2003

Regulation of translation initiation in mussels (Mytilus galloprovincialis, lam.) following contamination stress.

The use of mussels (Mytilus sp.) as bioindicators of the aquatic environment is a valuable approach to monitor environmental contamination. Applying batteries of biomarkers is an essential prerequisite, since the complexity of environmental contaminants can induce in mussels a variety of structural and functional responses, which are not necessarily correlated. In an attempt to correlate translation responses to contamination stress, the sedimentation profiles of runoff ribosomes isolated from digestive gland cells of control or contaminated Mediterranean mussels (Mytilus galloprovincialis, Lam.) were examined and the efficiency of these ribosomes to accomplish protein synthesis was determined. While the major species of ribosomal material was 80S monomers, native 60S and 40S ribosomal subunits were also detected independently of the contamination level in the surrounding waters. However, concomitant with the increase of contamination stress, the level of 80S ribosomes was reduced in favor of free ribosomal subunits. In addition, ribosomes isolated from contaminated mussels and programmed with poly(U) were less efficient to enzymatically bind AcPhe-tRNA, compared with ribosomes from control samples. These results suggest that the contamination stress causes stoichiometric aberrations in the ribosomal particle pool and reduction of translation machinery capability to initate protein synthesis. Data support the notion that downregulation of translation is an important component of the cellular stress response and may be exploited as a biomarker of environmental contamination.

Animals↗
PubMed1997

Rapid identification of heterotrophic, thermophilic, spore-forming bacteria isolated from hot composts.

The restriction enzyme profiles of 16S ribosomal DNAs (rDNAs) amplified by PCR from thermophilic heterotrophic bacterial strains isolated from composts were compared with those of reference strains. This allowed us to assign all but 1 of 16 strains to four different Bacillus species (namely, Bacillus stearothermophilus, Bacillus pallidus, Bacillus thermoglucosidasius, and "Bacillus thermodenitrificans"). This study showed that PCR restriction analysis of 16S rDNA contributes to rapid and reliable identification of newly isolated strains belonging to recognized species.

Bacillus↗
PubMed2001

Microorganisms with a taste for vanilla: microbial ecology of traditional Indonesian vanilla curing.

The microbial ecology of traditional postharvesting processing of vanilla beans (curing) was examined using a polyphasic approach consisting of conventional cultivation, substrate utilization-based and molecular identification of isolates, and cultivation-independent community profiling by 16S ribosomal DNA based PCR-denaturing gradient gel electrophoresis. At two different locations, a batch of curing beans was monitored. In both batches a major shift in microbial communities occurred after short-term scalding of the beans in hot water. Fungi and yeast disappeared, although regrowth of fungi occurred in one batch during a period in which process conditions were temporarily not optimal. Conventional plating showed that microbial communities consisting of thermophilic and thermotolerant bacilli (mainly closely related to Bacillus subtilis, B. licheniformis, and B. smithii) developed under the high temperatures (up to 65 degrees C) that were maintained for over a week after scalding. Only small changes in the communities of culturable bacteria occurred after this period. Molecular analysis revealed that a proportion of the microbial communities could not be cultured on conventional agar medium, especially during the high-temperature period. Large differences between both batches were observed in the numbers of microorganisms, in species composition, and in the enzymatic abilities of isolated bacteria. These large differences indicate that the effects of microbial activities on the development of vanilla flavor could be different for each batch of cured vanilla beans.

Bacillus↗
PubMed2002

Yeast diversity and persistence in botrytis-affected wine fermentations.

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.

Botrytis↗
PubMed2002

Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens.

A quantitative assay based on high-performance liquid chromatography analysis of bile salts and bacterial protein determination was established for investigating bile salt hydrolase (BSH) activity in bacteria isolated from the small intestine of chickens. Bacteria were isolated using various media and were subsequently grouped according to cell morphology, fermentation profile, and 16S ribosomal DNA sequence. Representative isolates from each bacterial group were assayed for BSH activity. The isolates differed in BSH activity with respect to the state of growth and preculturing with and without taurochenodeoxycholate. The highest levels of BSH activity were found with Enterococcus faecium and Clostridium perfringens.

Amidohydrolases↗
PubMed2005

Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-14C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 microM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 microg/ml, respectively. Mineralization of [U-14C]RDX to 14CO2 was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH4)2SO4- greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.

Actinomycetales↗
PubMed1998

Ultrastructural study in canine prostatic hyperplasia--effect of antiandrogen.

Ultrastructural changes in canine prostates after treatment with chlormadinone acetate (CMA) were investigated. Old male beagle dogs (5-8 years old ) were divided into two experimental groups; group 1 consisted of benign prostatic hyperplasia (BPH) controls, and group 2 received 0.3 mg/kg/day CMA orally for 6 months. In group 1 animals, the most striking ultrastructural changes were detected in the rough endoplasmic reticulum (rER) and Golgi complexes. The secretory granules were lined up along the apical plasma membrane, and exocytosis was frequently seen. In group 2 animals, the cytoplasm was electron-lucent and contained relatively few, poorly developed organelles. The rER was sparse and consisted of a few scattered, short profiles studded with ribosomes. The Golgi complexes were inconspicuous. The secretory granules were markedly decreased in both number and size. Furthermore, mitochondrial degeneration such as swollen or disappeared mitochondrial cristae or decreased electron density of the matrix were frequently seen in the smooth muscle cells. Based on our data, atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

Animals↗
PubMed1974

The structure of mononuclear phagocytes differentiating in vivo. I. Sequential fine and histologic studies of the effect of Bacillus Calmette-Guerin (BCG).

Although the differentiation of mononuclear phagocytes is fundamental to their multifarious activities, their differentiation is incompletely understood-particularly in vivo. The development of an epithelioid granuloma may be hypothesized to represent such differentiation in vivo. To test this, the sequential ultrastructure of developing epithelioid granulomas was examined. Viable bacilli Calmette-Guerin (BCG) injected into the subcutaneum of guinea pigs produced epithelioid granulomatous inflammation, which was sampled for light and electron microscopy on alternate days until the 33rd day after injection. Initially, monocytes invaded the tissues and then coalesced, enlarged and formed small granulomas which ultimately evolved into epithelioid granulomas. The monocytes, ultrastructurally very simple cells, developed increased nuclear euchromatin, prominent nucleoli, extensive cytoplasm, free ribosomes, abundant Golgi profiles, many mitochondria and numerous large lysosomes and became macrophages. The macrophages in turn underwent further enlargement and became closely intertwined with one another to form epithelioid cells-large polygonal macrophages, containing euchromatic nuclei, numerous lysosomes, plentiful mitochondria and prominent synthetic apparatus. These changes undergone by monocytes during their development into epithelioid cells, which may be divided into five stages, are interpreted as differentiation in vivo of the mononuclear phagocytes. The observations demonstrate directly the differentiation of these cells in vivo and suggest some, if not all, characteristic features of granulomatous inflammation result from such differentiation.

Animals↗
PubMed1971

Quantitative characterization of dense body, autophagic vacuole, and acid phosphatase-bearing particle populations during the early phases of glucagon-induced autophagy in rat liver.

Quantitative characterization of dense body, autophagic vacuole and acid phosphatase-bearing particle populations of rat liver have been made at 10 min intervals during the first 50 min following the intraperitoneal administration of glucagon. Beginning 10 to 20 min postinjection, increases in the number of autophagic vacuoles and in the osmotic sensitivity of acid phosphatase-bearing particles were observed, associated with a progressive disappearance of dense bodies. These changes appeared to reach a maximum 50 min after treatment. The average volume of autophagic vacuoles was found to be 440-870% greater than that of normal dense bodies during this time period. No consistent change in total acid phosphatase activity was noted. A detailed study of autophagic vacuole profile populations revealed the presence of five different types of profiles, two of which, types I and II, accounted for 76.3-94.4% of the profiles examined. Type I profiles primarily contained elements of the endoplasmic reticulum, free ribosomes, and ground cytoplasm. Type II profiles had mitochondrial profiles as their principal constituent, but endoplasmic reticulum and free ribosomes were also seen. At all time points type I profiles predominated, comprising 55-69% of the profiles found. Both profile types were bounded by single and double limiting membranes, the former being predominate. A time-dependent change in the ratio of single to double membrane-limited profiles could not be demonstrated. Morphometric parameters derived from profile size distributions indicated that the number of types I and II autophagic vacuoles increased with time, the rate being greater for the type II particle, except between 40 and 50 min. The average volume of the type II autophagic vacuole was consistently greater than that of the type I.

Acid Phosphatase↗
PubMed2001

Differentiation of Enterococcus spp. by cell membrane fatty acid methyl ester profiling, biotyping and ribotyping.

AIMS: Gas chromatographic analysis of cell membrane fatty acid methyl esters (FAME), biochemical profiling (biotyping) and EcoRI restriction endonuclease profiling of DNA containing ribosomal RNA sequences (ribotyping) were compared for differentiation of Enterococcus spp. METHODS AND RESULTS: FAME profiling, biotype profiling and ribotyping of 41 strains from retail Swiss-type cheeses and five strains from culture collections resulted in 17, 25 and 26 groups, respectively, with only two pairs of strains having the same FAME group, biotype profile and ribogroup. CONCLUSION: Substantial overlap occurred in groupings assigned by the three methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Differentiation of Enterococcus spp. strains increases if multiple methods are used.

Bacterial Typing Techniques↗
PubMed1986

[Comparative scanning and transmission electron microscopy studies of the ependyma of the central canal in the spinal cord of primates. I. Electron optical image of the ependyma in the central canal of the spinal cord of the callithrix monkey (Callithrix jacchus, Linné 1758)].

The ependyma lining the central canal of the spinal cord of adult males and females monkey, Callithrix jacchus, was examined by scanning and transmission electron microscopy. The cross section of the lumen of the central canal are round, oval, or triangular. Light and dark ependymal cells, depending on the density of the cytoplasm, were found. The light ependymal cells are fewer than the dark cells. The ependyma cytoplasm contained numerous mitochondria, filamentous structures, one or more well-developed Golgi-complexes, vesicles of the smooth endoplasmic reticulum, ribosomes, lysosomes, multivesicular bodies, profiles of the rough endoplasmic reticulum, large osmophilic bodies, and microtubules. The nuclei of the ependyma cells usually have a simple, regular round or oval shape. They occupy a relatively large portion of the cell volume and lie in the central or mediobasal position. Some of the nuclei show deep invaginations into the karyoplasm. Most of the mitochondria occupy mainly the supranuclear portion of the apical cytoplasm. There are of the crista-typ. Ribosomes occur free in the cytoplasm, but some attached to the profiles of the rough endoplasmic reticulum or being arranged as polysomes. The filamentous structures are generally prominent cytoplasmic components and are distributed at the apical, lateral, or basal region of the ependymocytes. They are grouped into bundles and arranged in parallel arrays. Some of these bundles reach the plasmamembrane at the free lumina of the central canal, others take contact to the filamentous structures of the zonulae adherentes of the junctional complex below the free surface. The granular endoplasmic reticulum shows specializations. There profiles surrounding granular substances and widely distributed granulations in connection with the nuclear envelope. The functional significance of the deposition of these granulations is still unknown. The luminal surface of the ependymocytes bears many microvilli and cilia. The cilia are regularly arranged in cranio-caudal direction. Each cilium has the typical (9 + 2)-subfibres. The intercellular space at the surface of the ependymal layer shows a single zonula adherens or zonulae adherentes in the row. Tight junctions and gap junctions were not found in the material examined. Cell processes of liquor contacting neurons between adjacent ependyma cells, protruding into the lumen of the central canal, could be observed. The termination of these neurons contains accumulations of mitochondria in the central part, large amounts of vesicles, and small dense bodies. They have short microvilli and some stereocilia at the free surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1969

Evidence for a proposed initiation complex for protein synthesis in reticulocyte polyribosome profiles.

Polysomes consisting of 40S ribosomal subunits attached to monosomes, disomes, trisomes, and tetrasomes have been isolated for the first time by the use of isokinetic sucrose density gradients. They are formed upon incubation of rabbit reticulocyte lysate in the presence of 10 mM NaF. It is proposed that these structures represent transient initiation complexes for protein synthesis.

Animals↗
PubMed1998

Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation.

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel, Spermophilus tridecemlineatus. PS, in vivo, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37 degreesC, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 +/- 0.08 pmol/mg protein per min; hibernator, 0.16 +/- 0.05 pmol/mg protein per min, P < 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2alpha are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 +/- 0.7 min; hibernator, 7.1 +/- 1.4 min, P < 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate "trickle" blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.

Adenosine Diphosphate Ribose↗
PubMed1976

Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis.

1. Messenger activity of phage f2 RNA modified with methoxyamine under non-denaturing conditions was studied in E. coli-free system. The incorporation of amino acids into phage polypeptides was decreased, and the synthesis of phage-specific proteins was diminished. The RNA replicase synthesis was more affected than synthesis of coat protein. The impaired messenger activity of the methoxyamine-modified f2 RNA was due to the blocking of elongation process by modified cytosines present in RNA chain. 2. Specificity of f2 RNA to stimulate ribosomal binding predominantly at the coat protein initiation site was not affected by methoxyamine-treatment, as demonstrated by unchanged binding of f[3H]Met-tRNA and [14C]alanyl-tRNA to ribosomes. 3. Unfolding of f2 RNA molecule on treatment with methoxyamine in the presence of guanidine-HCl resulted in a significant increase of RNA capacity to direct fMet-tRNA binding to ribosomes. Sucrose-density gradient profiles revealed the formation of polysome-like initiation complexes indicating that ribosomes were able to bind at many hitherto inaccessible initiation codons in RNA molecules. fMet-tRNA bound to ribosomes in the presence of unfolded RNA was found to be fully reactive with puromycin.

Cell-Free System↗