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.

Use of the 'ex vivo' test to study long-term bacterial survival on human skin and their sensitivity to antisepsis.

AIMS: To determine bacterial survival on human skin and their sensitivity to antisepsis. METHODS AND RESULTS: An 'ex vivo' protocol which uses human skin samples placed into diffusion cells, and electron microscopy (EM), were used to study the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa inoculated onto skin samples over a 46-h incubation period at 32 degrees C. Concurrently variation in skin pH was evaluated at different time intervals during this period. In addition the antimicrobial activity of three antiseptics against the incubated micro-organisms was assessed quantitatively with the 'ex vivo' test, while their detrimental effects against bacteria were observed by EM. All three bacteria were still present in high number after 46 h inoculation on skin, although the concentration of E. coli and S. aureus were reduced by 2.74 and 1.58 log(10) reduction, respectively, over this period of time. Electron micrographs showed clear evidence of cell division and some bacteria appeared to be embedded into the skin layers. The antiseptics tested had some antibacterial activity against bacteria incubated on skin for 3 and 10 h, and EM evidence showed some morphological damages including cellular blebbing and the presence of fibrillar material around the cells. All micro-organisms had an acidifying effect on skin samples. CONCLUSIONS: Here, it was shown that bacterial pathogens can survive and grow when incubated on human skin. In addition, it is possible that they can penetrate the stratum corneum, which can provide some protection against antisepsis. SIGNIFICANCE AND IMPACT OF THE STUDY: The apparent low bactericidal activity of biocides attributed in part to bacterial protection from skin layers is particularly important to assess in order to ensure antisepsis efficacy.

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.

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.

Assessment of skin viability: is it necessary to use different methodologies?

BACKGROUND/AIM: Skin is complex and may display variable structural and metabolic change 'ex vivo'. The present study aimed to follow measures of skin viability and evaluate their usefulness as markers of viability. MATERIALS AND METHODS: We evaluated the viability of skin samples fresh or after being frozen and subsequently thawed. Assessments included histopathological appearance, lactate dehydrogenase (LDH) activity, oxygen consumption and skin pH. RESULTS: Morphological investigations of fresh and frozen skin samples using light and electron microscopy showed samples with relatively well-defined epidermis and dermis. Frozen samples showed some sign of stratum corneum fragmentation, although this was not obvious. LDH activity measured in fresh samples kept at 4 degrees C was low, but it was stable up to 7 days. Fresh samples kept at 32 degrees C had a comparable LDH activity to the ones kept in the fridge up to 4 days. Frozen samples, thawed and then kept at 4 degrees C showed a stable LDH activity after 24 h of incubation. However, frozen samples incubated at 32 degrees C demonstrated a high variability in results, with up to 800 U/L of LDH activity after 5 days of incubation. Freshly excised as well as freshly thawed samples showed the highest respiration rates. Fresh and thawed samples stored for a long period of time had a significantly lower (sometimes non-existent) oxygen consumption rate. Our results also showed an increase in the oxygen consumption rate of fresh samples being incubated at 32 degrees C for 24 h. The oxygen consumption rate for all samples reached a plateau within the 15-min measurement period and even the fresh samples did not deplete all the oxygen from the medium. Skin samples ex vivo showed a significantly higher pH than human skin in vivo, and when incubated for 46 h at 32 degrees C, fresh samples had a significantly lower pH than frozen samples. All protocols were reproducible and freshly excised and freshly thawed skin samples showed the highest rates of viability. CONCLUSION: ex vivo skin shows variation of several parameters over time. It is recommended to use two or three techniques for evaluation of skin viability including at least oxygen measurement and an enzyme assay.

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.

Effects of ortho-phthalaldehyde, glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability.

The mechanisms of the mycobactericidal action of ortho-phthalaldehyde (OPA), glutaraldehyde (GTA) and chlorhexidine diacetate (CHA) were investigated using mycobacterial spheroplasts of two reference strains, Mycobacterium chelonae NCTC 946, Mycobacterium abscessus NCTC 10882 and two GTA-resistant strains, M. chelonae Epping and M. chelonae Harefield. Transmission electron microscopy of the spheroplasts revealed an altered cell wall structure compared with the parent cells. Structural alterations resulting from the spheroplasting process were in part correlated to a loss of lipid content. Low concentrations of CHA induced protein coagulation in M. chelonae NCTC 946 spheroplasts, which also exhibited the highest loss of free non-polar lipids. Higher concentrations of CHA were required to produce similar results to the other spheroplasts investigated in which there was a less substantial decrease in lipid content. OPA (0.5% w/v) readily penetrated the residual cell wall and cytoplasmic membrane, producing significant protein coagulation in M. chelonae NCTC 946. GTA (0.5% v/v) induced a similar effect but to a lesser extent. Pre-treatment of the spheroplasts with OPA and GTA and their subsequent suspension in water demonstrated that GTA was a more potent cross-linking agent. This protective effect of GTA results from extensive cross-linking of amino and/or sulphydryl side-chain groups of proteins. The rapid mycobactericidal effect of OPA probably arises from its more efficient penetration across biological membranes. Mycobacterial spheroplasts represented a useful cellular model with an altered cell wall permeability. This study also showed the importance of the mycobacterial cell wall in conferring intrinsic resistance to CHA.

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.

Possible mechanisms for the relative efficacies of ortho-phthalaldehyde and glutaraldehyde against glutaraldehyde-resistant Mycobacterium chelonae.

AIMS: This investigation compared glutaraldehyde (GTA)-sensitive and -resistant strains of Mycobacterium chelonae and examined the effects of pretreatment of GTA-sensitive and -resistant strains of Myco. chelonae with chemical agents that interfere with cell wall synthesis. METHODS AND RESULTS: When exposed to 2% (v/v) GTA at 25 degrees C, GTA-resistant strains of Myco. chelonae dried on to glass carriers were not inactivated to any significant extent. By contrast, GTA-sensitive strains of Myco. chelonae and a strain of Myco. terrae suffered a > 6 log reduction in viability in 5 min. However, ortho-phthalaldehyde (OPA; 0.5% w/v) achieved a corresponding inactivation against two GTA-resistant strains within 5-10 and 10-20 min, respectively. Electron microscopy, using a non-aldehyde fixation process and also negative staining, failed to detect any extensive changes in GTA-sensitive and -resistant cultures exposed to GTA or OPA. Thin-layer chromatography was unsuccessful in detecting differences between GTA-resistant and -sensitive strains of Myco. chelonae. However, pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility further to OPA but not to GTA. CONCLUSION: Ortho-phthalaldehyde is an effective new biocidal agent that, at its in-use concentration, is rapidly bactericidal to non-sporulating bacteria, including GTA-sensitive and -resistant mycobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility to OPA but not to GTA.

Encystation in Acanthamoeba castellanii: development of biocide resistance.

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.

Cytological changes in chlorhexidine-resistant isolates of Pseudomonas stutzeri.

Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive analysis of X-ray (EDAX) have been used to examine chlorhexidine diacetate (CHA)-sensitive and -resistant isolates of Pseudomonas stutzeri and to determine the effects of CHA on the cells. Significant differences were observed in the structure, size and elemental composition of CHA-sensitive and -resistant cells. Treatment with CHA produced considerably greater changes in CHA-sensitive cells, with widespread peeling of the outer membrane, a substantial loss of cytoplasmic electron-dense material and extensive lysis. Cells from the resistant isolates showed no blebbing of the outer membrane and no structural damage. X-ray mapping confirmed the difference in CHA uptake between CHA-sensitive and CHA-resistant cells. It is proposed that changes in the outer membrane form a major mechanism of resistance to CHA in P. stutzeri.

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.

X-ray microanalysis of chlorine and phosphorus content in biguanide-treated Acanthamoeba castellanii.

Energy dispersive analysis of X-rays (EDAX) was used to study the effects of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) on Acanthamoeba castellanii. A high variation of elements occurred in untreated individual cells and only two elements, Cl (a biocide marker) and P, were investigated. X-ray dot mapping of untreated trophozoites and cysts revealed that Cl in cells was uniformly distributed throughout the cytoplasm, whereas P was less dense in the vacuoles. X-ray dots of Cl in biocide-treated trophozoites and cysts appeared denser and evenly distributed within the cells as the biguanide concentration increased. Quantitative analysis of either CHA or PHMB within the cells using Cl as an elemental marker was unsatisfactory because of the high Cl levels in untreated cells. The apparent increases of P in some experiments with treated cells might be associated with reduced permeability, protein coagulation or aggregation of phospholipids.

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.

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.

Efficacy and mechanisms of action of sodium hypochlorite on Pseudomonas aeruginosa PAO1 phage F116.

The Pseudomonas aeruginosa PAO1 phage F116 was used to investigate the viricidal activity and the mechanism of action of sodium hypochlorite. The bacteriophage was inactivated with a low concentration (0.0005% available chlorine) of the biocide prepared in tap water but it was less sensitive to a sodium hypochlorite solution prepared in ultra-pure water (0.0075% available chlorine). For all the effective concentrations of sodium hypochlorite (i.e. producing at least 4 log reduction in phage titre), F116 was readily inactivated within 30 s. Electron microscopical investigations of the phage particles challenged with sodium hypochlorite showed a wide variety of deleterious effects, some of which have not been previously observed with other biocides. The wide range of structural alterations observed suggested that sodium hypochlorite has multiple target sites against F116 bacteriophage. A 30 s exposure to sodium hypochlorite (0.001% available chlorine) produced severe damage, the number and severity of which increased with a higher concentration (0.0075% available chlorine) and with a longer contact time. These observations suggested that sodium hypochlorite inactivated F116 bacteriophage by causing structural alterations to the phage head, tail and overall structure, hence possibly releasing the viral genome from damaged capsids in the surrounding media.

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study.

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 microg ml(-1) produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.

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.

Non-variable sources of pure water and the germination and outgrowth of Bacillus subtilis spores.

Variable germination and outgrowth occurred when Bacillus subtilis NCTC 8236 spores were inoculated into nutrient broth prepared with distilled water. More reproducible findings were achieved when the medium was prepared with Elgastat water and the greatest reproducibility occurred with Elgastat water as vehicle combined with a rigorous acid-washing of all glassware. This combined procedure also produced optimum and reproducible results for the synchronous growth of two B. subtilis 168 strains in casein medium supplemented with appropriate amino acids, a technique of value in monitoring the development of resistance to antibacterial agents during sporulation. The levels of aluminium in distilled water were higher than those of other elements; however, the incorporation of aluminium sulphate into broth prepared with Elgastat water had no effect on germination, and outgrowth was reduced (but not eliminated) only at high concentrations of this salt.

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.