Glycogen autophagy in the liver and heart of newborn rats. The effects of glucagon, adrenalin or rapamycin.

The effects of glucagon, adrenalin or rapamycin on glycogen autophagy in the liver and heart of newborn rats were studied using biochemical determinations and electron microscopy. Glucagon or adrenalin increased autophagic activity in the hepatocytes and myocardiocytes, glycogen-hydrolyzing acid glucosidase activity in the liver and heart and degradation of glycogen inside the autophagic vacuoles. Glucagon or adrenalin also increased the maltose-hydrolyzing acid glucosidase activity in the liver, but not in the heart. Similar effects were produced in the newborn heart by rapamycin. These observations support previous studies suggesting that the cellular machinery which controls glycogen autophagy in the liver and heart of newborn animals, is regulated by the cyclic AMP and the mTOR pathways.

Simulation of vascular surfaces: differential saturation with glycosaminoglycans and their quantitative analysis.

Surface layers of sulphated glycosaminoglycan can be quantified by X-ray microanalysis using the local mass-fraction of the element sulphur. As a calibration standard radiolabelled chondroitin sulphate has been attached covalently to a nylon surface at various densities to the point where the molecules are packed as close as the radius of gyration permits.

An electron microscopic and biochemical study of the effects of propranolol on the glycogen autophagy in newborn rat hepatocytes.

The effects of propranolol on the glycogen autophagy in newborn rat hepatocytes were studied by using biochemical determinations, electron microscopy and morphometric analysis. Propranolol lowered the liver cyclic AMP and cyclic AMP-dependent protein kinase activity. It also decreased the formyl-methionyl-leucyl-phenylalanine (FMLP)-inhibitable Ca2+-ATPase activity including lysosomal calcium uptake pump. The normal postnatal increase in the volume of autophagic vacuoles and the activity of acid glycogen-hydrolyzing alpha glucosidase were inhibited. Also, the degradation of glycogen inside the autophagic vacuoles was apparently inhibited. The activity of acid mannose 6-phosphatase was increased. These findings indicate that propranolol influences several steps in the sequence of events leading to the breakdown of glycogen in the autophagic vacuoles of newborn rat hepatocytes. This supports our previous studies suggesting that cyclic AMP regulates glycogen autophagy.

Studies on glycogen autophagy: effects of phorbol myristate acetate, ionophore A23187, or phentolamine.

The effects of agents that could manipulate the lysosomal calcium such as phorbol myristate acetate, ionophore A23187, and phentolamine on the lysosomal glycogen degradation were studied by electron microscopy, morphometric analysis, and biochemical assays in newborn rat hepatocytes. Phorbol myristate acetate, which promotes the input of calcium to lysosomes, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid alpha 1,4 glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles and also decreased the activity of acid mannose 6-phosphatase. Ionophore A23187, which releases lysosomal calcium, produced opposite results in these enzyme activities. Phentolamine, an alpha-adrenergic blocking agent which interferes with the generation of phosphoinositides and may activate the lysosomal calcium uptake pump, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles. The results of this study constitute evidence that changes in lysosomal calcium may influence certain aspects of autophagy, including the degradation of glycogen inside the autophagic vacuoles. They also support our previous postulate [Kalamidas and Kotoulas (2000a,b) Histol Histopathol 15:29-35, 1011-1018] that stimulation of autophagic mechanisms in newborn rat hepatocytes may be associated with acid mannose 6-phosphatase activity-deficient lysosomes.

Electron microscopic and biochemical study of the effects of rapamycin on glycogen autophagy in the newborn rat liver.

The effects of rapamycin on glycogen autophagy in the newborn rat liver were studied using biochemical determinations, electron microscopy, and morphometric analysis. Rapamycin increased the fractional volume of hepatocytic autophagic vacuoles, the liver lysosomal glycogen-hydrolyzing activity of acid glucosidase, the degradation of glycogen inside the autophagic vacuoles, and decreased the activity of acid mannose 6-phosphatase. These findings suggest that rapamycin, a known inhibitor of the mammalian target of rapamycin (mTOR) signaling, induces glycogen autophagy in the newborn rat hepatocytes. mTOR may participate in the regulation of this process.

Electronmicroscopic investigation of the effects of biocides on Pseudomonas aeruginosa PAO bacteriophage F116.

Electronmicroscopy was used to observe morphological changes of the Pseudomonas aeruginosa PA0 bacteriophage F116 when treated with various biocides commonly used as antibacterial and antifungal agents. Because of its large size (145 nm) and its organised structure (an isometric head and a tail), it was possible to classify structural damage into eight categories. The morphological changes induced depended on the type of biocide used and its concentration. Glutaraldehyde increased the number of phages with empty heads. Peracetic acid and phenol altered the appearance of the viral genome packaged inside the head, produced fractured heads, and damaged the tail. Peracetic acid also induced folding of the phage heads. The alcohols tested also altered the head structure. Cetylpyridinium chloride induced mainly fractured head damage. Chlorhexidine had little effect on the structure of F116.

Structural changes induced by mupirocin in Staphylococcus aureus cells.

Cells of mupirocin-sensitive, moderately-resistant and highly-resistant cultures of Staphylococcus aureus (mupirocin MICs 0.13, 16 and > 512 mg/l, respectively) were exposed to various concentrations of the antibiotic and examined by transmission electron microscopy. The most severe damage occurred in mupirocin-sensitive cells. Cells from moderately-resistant cultures trained in vitro to high-level mupirocin resistance were more hydrophobic than the parent cells. The antibiotic was slowly lethal to the mupirocin-sensitive strain and sub-inhibitory concentrations prevented or reduced growth of the other strains over a 6 h incubation period, irrespective of whether the drug was added at zero time or in the exponential growth phase.

The growth of Salmonella typhimurium on irradiated, raw, skinless chicken breast.

Salmonella typhimurium was inoculated onto 1 g cubed samples of irradiated, raw, skinless chicken breast, which were then incubated at 30 degrees C under humid conditions. Kinetic growth data was obtained by means of viable counts performed on triplicate samples over a 24 h period. In addition, the spatial arrangement of cells on samples taken 6, 12 and 24 h after inoculation was observed by scanning electron microscopy. The population entered exponential growth approximately 3 h after inoculation, and maintained a constant rate of growth for approximately 13 h before entering a stationary phase. A generation time of 0.74 h was recorded. Scanning electron microscopy observations revealed colonial development from loose clusters of cells at 6 h to discrete, compact microcolonies (approximately 40 microns in diameter) by 12 h. By 24 h colonies were well-developed (approximately 600-700 microns in diameter) with a well-defined colony periphery. The results of this study give insight into the growth and development of bacteria on meat tissue, and serve to highlight that the nature of such growth is quite different from that in dispersed liquid culture systems, i.e. those traditionally used to model such growth.

Chondroitin sulphate at the endothelial lumen of pig aorta: assay by radiolabelling and by X-ray microanalysis.

Trypsin-releasable glycosaminoglycans from the luminal surface of intact pig aorta were measured following metabolic labelling with [35S]sulphate. Chondroitin sulphate was found to be present at a surface density equal to that already established for heparan sulphate (5 X 10(11) chains per cm2). This result was confirmed by X-ray microanalysis of the luminal sulphur content before and after treatment with specific glycosaminoglycan-degrading enzymes. This result implies that approximately half of the luminal surface is occupied by sulphated glycosaminoglycans.

Putrescine uptake by alveolar epithelial cell monolayers exhibiting differing transepithelial electrical resistances.

Rat alveolar type II cells were isolated following elastase digestion and cultured on polycarbonate filters at various densities and in different media. Two days after seeding, the cells formed a monolayer on the filters which consisted predominantly of type II cells, these then de-differentiated to a alveolar type I-like cell monolayer by day 6. The seeding density and media utilized affected the transepithelial electrical resistance (TEER) generated by the monolayer. Only certain culture conditions allowed the production of a monolayer that mimics, putatively, the in vivo alveolar epithelium (TEER greater than 1000 omega cm2). Vmax and K(m) values for the uptake of putrescine by monolayers exhibiting low and high TEERs on day 6 were determined. The capacity of the putrescine uptake mechanisms was greater in cell monolayers exhibiting a high TEER than those exhibiting a low TEER, suggesting that the TEER does not only measure the "tightness" of the monolayer but contains an element representative of the viability of the cell monolayer. The selection of appropriate TEERs for cell culture investigations is discussed.

An in vitro approach for the study of dentinogenesis by organ culture of the dentine-pulp complex from rat incisor teeth.

Culture of the developing dental tissues has contributed to understanding of developmental processes during early odontogenesis. However, to understand fully the mechanisms involved during dentinogenesis and tissue repair there is a need to develop culture models for the dentine-pulp complex from more mature dental tissues. This study describes the development of a system for the organ culture of mature rodent teeth. Slices of incisors from 28-day-old rats were embedded in a semisolid, agar-based medium and cultured on floating Millipore filters at the liquid-gas interface for up to 14 days. Preservation of cell and tissue morphology was observed throughout the entire dentine-pulp complex after each culture period and autoradiographic studies showed that the odontoblasts were actively synthesizing and secreting extracellular matrix during culture. Transmission electron microscopy confirmed that the phenotypic morphology of the odontoblasts had been maintained during culture. These results demonstrate that the dentine-pulp complex from mature rodent tissues can be cultured successfully for substantial periods of time and will provide a useful model for the study of dentinogenesis and tissue repair.

Histochemical localization of acid mannose 6-phosphatase activity in the newborn rat hepatocytes.

The localization of acid mannose 6-phosphatase activity in newborn rat hepatocytes was demonstrated at the electron microscopic level by using a histochemical method based on the work of Robinson and Karnovsky. Reaction product was virtually restricted to the lysosomes. Most of them exhibited various grades of reactivity. Some were devoid of activity. Our observations suggested that this histochemical method could be used to differentiate distinct subpopulations of lysosomes on the basis of their acid mannose 6-phosphatase activity.

Quantitative X-ray microanalysis of adrenal medullary cells of young adult and aged rats after glutaraldehyde fixation and potassium dichromate treatment.

X-ray microanalysis has been used to detect chromium in the histochemical reaction product resulting from the reaction of noradrenaline with glutaraldehyde during fixation of the rat adrenal medulla and subsequent treatment with potassium dichromate. In unstained ultrathin sections, noradrenaline cells can be identified by their content of highly electron-dense storage granules, which enables individual granules to be analysed quantitatively to assess the amount of bound chromium within them. In young adult (4-month-old) rats the mean chromium content of noradrenaline-containing adrenal medullary granules was 443.6 +/- 50.7 mM/kg dry weight. In aged (24-month-old) animals the mean chromium content was 267.0 +/- 64.0 mM/kg dry weight which was significantly (P < 0.01) lower then the value for the young adult rats. Some noradrenaline cells contained granule populations, which were markedly less electron dense than those in the young adults and this is reflected in the ranges of chromium values recorded between individual cells in the 24-month-old animals. There were also noradrenaline cells in the medulla of the aged animals, which contained highly electron-dense granules but these did not contain as much bound chromium as the highest values recorded in the young adult animals. The results are discussed in the context of the growth of the rat adrenal medulla throughout the lifespan and with respect to the effects of age on the integrity of storage granules.

Degradative inactivation of the peroxisomal enzyme, alcohol oxidase, during adaptation of methanol-grown Candida boidinii to ethanol.

Adaptation of methanol-grown C. boidinii to ethanol-utilization in non-growing cells resulted in decreased activity of the peroxisomal enzyme alcohol oxidase. Re-appearance of alcohol oxidase activity was dependent on protein synthesis de novo. Degradation of alcohol oxidase protein was shown to parallel the decrease in activity. Adaptation of methanol-grown cells to ethanol-utilization resulted in increased absorbance due to cytochromes and decreased absorbance due to flavoprotein. Decrease in alcohol oxidase activity was associated with loss of the flavin coenzyme, FAD, from the organisms and the appearance of flavins (FAD, FMN, riboflavin) in the surrounding medium. Electron microscopic observations showed that general degradation of whole peroxisomes rather than specific loss of crystalline cores (alcohol oxidase protein) occurred during the adaptation.

Characterization of bacteria by multiparameter flow cytometry.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.

A note on the development of a non-aldehyde fixation technique for examining spores of Bacillus subtilis under the electron microscope.

Various techniques were studied for fixing spores of Bacillus subtilis prior to examining them by transmission or scanning electron microscopy. A non-aldehyde technique employing carbodiimide in cacodylate buffer produced excellent results and could be of value in studying the cytological changes produced in spores exposed to inimical treatments.

Resistance of Pseudomonas aeruginosa PAO1 phage F116 to sodium hypochlorite.

The development of viral resistance to sodium hypochlorite was investigated using the Pseudomonas aeruginosa bacteriophage F116 as a model system. This phage was chosen because of its structural characteristics and former investigations conducted in this laboratory. F116 was shown to be sensitive to a sodium hypochlorite concentration of 0.0075 gl-1 (available chlorine) which produced a 5 log10 reduction in titre in a suspension test. Survival bacteriophages challenged with this sodium hypochlorite concentration were isolated, propagated and challenged again with the same and higher concentrations of the biocide. It was observed that progeny virions were becoming increasingly resistant to sodium hypochlorite challenges up to a concentration of 0.0175 gl-1 of available chlorine. It was also noticed that 1-2 log10 of F116 virions from resistant phage lysates remained sensitive to the biocide. An electron microscopical investigation of F116 resistant lysates showed that the phage resistance to sodium hypochlorite was not caused by F116 particles aggregation. Furthermore, no morphological difference between the sensitive and resistant F116 particles to sodium hypochlorite was identified.

Energy-dispersive X-ray microanalysis of membrane-associated inclusions in hydrogenosomes isolated from Trichomonas vaginalis.

Energy-dispersive X-ray microprobe analysis of electron-dense inclusions in hydrogenosomes isolated from the aerotolerant anaerobic protozoal human parasite Trichomonas vaginalis, Bushby strain, indicated the presence of high levels of Mg, P and Ca. This suggested that divalent cation (e.g. Ca2+ or Mg2+) accumulation by hydrogenosomes may be important in the regulation of intracellular ion concentrations.

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

This paper reports for the first time the presence in the anaerobic rumen ciliate Dasytricha ruminantium (Schuberg) of microbody-like organelles, about 0.5 micrometer diameter, with a granular matrix and an equilibrium density of approx. 1.18 g/ml. These organelles can be isolated in a fraction sedimented at 10(5) g-min that contains 67% of the total pyruvate synthase (EC 1.2.7.1), 66% of the hydrogenase (EC 1.18.3.1) and 20% of the lactate dehydrogenase (EC 1.1.1.27). Thus in several respects this fraction is enzymically similar to those containing hydrogenosomes in some other parasitic anaerobic protozoa (the trichomonads). However, in contrast with the hydrogenosomes of trichomonads, the oxygen-tolerant enzyme malate dehydrogenase (decarboxylating) (EC 1.1.1.40) is not particulate, but occurs only in the cytosol. These results enable the proposal of a scheme for the pathway of product formation (acetate, lactate, CO2 and H2) from carbohydrates.

Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.

When Azotobacter vinelandii is grown under nitrogen-fixing conditions, the mean cell volume fluctuates from 2.7 to 6.6 microns 3 as determined using a Coulter counter. When NH4Cl is supplied as nitrogen source, the mean cell volume fluctuates from 4.6 to 7.4 microns3. Parallel experiments using flow cytometric measurements show similar characteristic fluctuations in the narrow forward angle light scattering signal and also in cellular protein content as determined using fluorescein isothiocyanate (FITC) fluorescence. Fluctuations in the perpendicular light scatter signal during batch growth are similar for both sets of growth conditions. Changes in cell morphology and ultrastructure are also similar for both sets of growth conditions, as demonstrated by electron microscopic examination. We conclude that narrow forward angle light scatter is a close correlate of cell size, whereas right angle scatter is an indicator of morphological variations other than size.