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PubMed1983

Extracellular slime associated with Proteus mirabilis during swarming.

Light microscopy, transmission electron microscopy, and scanning electron microscopy were used to visualize the extracellular slime of Proteus mirabilis swarm cells. Slime was observed with phase-contrast microscopy after fixation in hot sulfuric acid-sodium borate. Ruthenium red was used to stain slime for transmission electron microscopy. Copious quantities of extracellular slime were observed surrounding swarm cells; the slime appeared to provide a matrix through which the cells could migrate. Swarm cells were always found embedded in slime. These observations support the argument that swarming of P. mirabilis is associated with the production of large quantities of extracellular slime. Examination of nonswarming mutants of P. mirabilis revealed that a number of morphological changes, including cell elongation and increased flagellum synthesis, were required for swarm cell migration. It is still unclear whether extracellular slime production also is required for migration.

Flagella↗
PubMedDate not supplied

[Nitrogen fixing activity of pea nodule bacteria during different phases of host plant development].

The activity of nitrogenase in nodules of pea plants and the rate of diurnal nitrogen fixation were studied at different phases of the plant growth using the acetylene and isotope techniques. At the same time, the morphology of nodule bacteria was studied by electron and phase-contrast microscopy. About 90% of the bacteria in nodules were found in the form of bacteroids from the early phase of the plant growth (budding) to ripening when gemmated arthrospores could be observed on some bacteroids. The highest activity of nitrogenase and nitrogen fixation was detected at the reproductive stage of growth. Therefore, in order to obtain more active strains of pea nodule bacteria, they should be isolated from nodules at this stage of plant growth.

Fabaceae↗
PubMed1977

Morphology of mouse epidermal cells in vitro: a scanning electron microscopy study.

Scanning electron microscopy of isolated epidermal cells from newborn mice grown in vitro showed that the cultures consisted of several morphologically different types of cells. Young cultures had many smooth, round cells while older cultures contained more cells with rough or ruffled surface and a varying number of flat, irregular cells. Probably, the various cell types corresponded to different stages of differentation (keratinization). Cultures that were grown in the presence of retinyl acetate (12.5 microgram/ml) had more round and smooth cells after several days in vitro than the controls. This could indicate that retinyl acetate delayed or altered cell differentiation. Scanning electron microscopy of the cultures consistently showed that the cells were situated at different layers. The apparent monolayer seen by phase contrast microscopy therefore seems to be an optical phenomenon due to projection of the cells onto the same plane.

Animals↗
PubMed2001

Provider-performed microscopy.

The category of provider-performed microscopy was defined in the Federal Register to provide a unique regulatory approach for bright-field and phase-contrast microscopy performed by a physician, dentist, or midlevel practitioner examining labile specimens. A CLIA certificate for this category of testing is available, which also permits the performance of waived tests. Because these provider-performed microscopy procedures do not have quality-control materials available, special challenges are encountered in establishing and monitoring such testing within a hospital. Vigilance is required to maintain a provider-performed microscopy program within a hospital.

Adult↗
PubMed1979

Normal and pseudorabies virus infected primary nerve cell cultures in scanning electron microscopy.

Primary cell cultures from the central nervous system of the embryonic rat were inoculated with pseudorabies virus. Their morphological changes were studied by phase contrast microscopy and by scanning as well as by transmission electron microscopy. Uninfected cultures display two distinct cell layers in scanning electron microscopy: a flat continuous monolayer supports a heterogeneous population of individual, presumably neural cells, which emit processes of different number and size. The latter cells form contacts by a dense network of fibres. Infectious virus is propagated in these nerve cell cultures with similar effectivity as in other cultures. The infectoin leads to fusion and death of the cells. By the time the cytopathic effect is visible, nearly all cells, including those of neuronal and those of nonneuronal appearance, are studded with ample amounts of virus-sized particles. The particles represent viruses as demonstrated by transmission electron microscopy or by treatment with a hyperimmune serum directed against pseudorabies virus structural components. Hyperimmune serum leads to clustering of the particles at the cell surface. The amount of virus particles per surface unit was about 10 times higher on neural cells as compared to primary rabbit kidney cells. The concentration of infectious particles in the supernatant, however was approximately the same. The system described appears to be useful for the study of acute virus effects on neural tissue under strictly controlled conditions.

Animals↗
PubMed2001

Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy.

Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-micron-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation.

Blood Platelets↗
PubMed2005

Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system.

BACKGROUND: The demand for the clinical use of hepatocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes. METHODS: The domestic extracorporeal circulating perfusion apparatus was used to isolate and harvest swine hepatocytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and observed by the trypan blue exclusion test, biochemical measurements, phase contrast microscopy and transmission electron micrography (TEM). RESULTS: The total yield of isolated hepatocytes reached to 1.5(+/-0.4)X10(10) per liver with a viability of 92(+/-5)%, and the purity of hepatocytes reached to 98%. Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly. CONCLUSION: This method of procuring swine hepatocytes could get high quality cells with active metabolic function.

Animals↗
PubMed1989

Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen.

Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H2:CO2 (80:20) or N2:CO2 (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H2:CO2. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H2:CO2 to form acetate in quantities 2 to 8-fold higher than when grown under N2:CO2. All presumptive acetogenic isolates were derived from either the 10(-7) or 10(-8) dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contrast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species.

Acetates↗
PubMed1989

Expression of two gas vacuole protein genes in Halobacterium halobium and other related species.

The archaebacterium Halobacterium halobium contains two genes encoding gas vacuole proteins (vac). One resides on a large naturally occurring plasmid and encodes a protein of 76 amino acids (p-vac), while the other is a chromosomal gene that encodes a highly similar protein of 79 amino acids (c-vac). Northern analysis determined the c-vac and p-vac mRNA to be approximately 340 nucleotides in length, and S1 mapping of both transcripts indicated that the 5' terminus for each starts at the same relative nucleotide. Three other Halobacterium species producing gas vacuoles were investigated, H. spec. GN101, YC819-9, and SB3. All three contain only a chromosomal c-vac gene, and the 5' terminus of the 340 nucleotide mRNA starts at the same nucleotide as found for H. halobium. The c-vac gene region of H. spec. GN101 contains nine nucleotide exchanges, three of which occur in the coding region with no effect on the amino acid sequence. In contrast, the c-vac gene of H. spec. SB3 has an identical nucleotide sequence to the H. halobium c-vac gene. Gas vacuole production in each of these species was monitored during culture growth by phase contrast microscopy, and the vac mRNA level was determined for each time point. H. halobium p-vac deletion mutants, as well as the halobacterial species GN101 and YC819-9, start to synthesize gas vacuoles in early stationary growth phase with a maximal mRNA content in stationary phase. In contrast, H. halobium wild-type synthesizes gas vacuoles exclusively due to p-vac gene expression with a maximal mRNA level during logarithmic growth, and transcripts of the c-vac gene were not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaeal Proteins↗
PubMed2004

Shape changes and vesicle fission of giant unilamellar vesicles of liquid-ordered phase membrane induced by lysophosphatidylcholine.

Liquid-ordered phase (lo phase) of lipid membranes has properties that are intermediate between those of liquid-crystalline phase and those of gel phase and has attracted much attention in both biological and biophysical aspects. Rafts in the lo phase in biomembranes play important roles in cell function of mammalian cells such as signal transduction. In this report, we have prepared giant unilamellar vesicles (GUVs) of lipid membranes in the lo phase and investigated their physical properties using phase-contrast microscopy and fluorescence microscopy. GUVs of dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol membranes and also GUVs of sphingomyelin (SM)/cholesterol membranes in the lo phase in water were formed at 20-37 degrees C successfully, when these membranes contained >/=30 mol % cholesterol. The diameters of GUVs of DPPC/cholesterol and SM/cholesterol membranes did not change from 50 to 28 degrees C, supporting that the membranes of these GUVs were in the lo phase. To elucidate the interaction of a substance with a long hydrocarbon chain with the lo phase membrane, we investigated the interaction of low concentrations (less than critical micelle concentration) of lysophosphatidylcholine (lyso-PC) with DPPC/cholesterol GUVs and SM/cholesterol GUVs in the lo phase. We found that lyso-PC induced several shape changes and vesicle fission of these GUVs above their threshold concentrations in water. The analysis of these shape changes indicates that lyso-PC can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. Threshold concentrations of lyso-PC in water to induce the shape changes and vesicle fission increased greatly with a decrease in chain length of lyso-PC. Thermodynamic analysis of this result indicates that shape changes and vesicle fission occur at threshold concentrations of lyso-PC in the membrane. These new findings on GUVs of the lo phase membranes indicate that substances with a long hydrocarbon chain such as lyso-PC can enter into the lo phase membrane and also the raft in the cell membrane. We have also proposed a mechanism for the lyso-PC-induced vesicle fission of GUVs.

Lysophosphatidylcholines↗
PubMed1998

Measurement of motility of Helicobacter pylori, Campylobacter jejuni, and Escherichia coli by real time computer tracking using the Hobson BacTracker.

AIMS: (1) To make precise measurements and comparisons of various aspects of motility of three gastrointestinal pathogens, Helicobacter pylori, Campylobacter jejuni, and Escherichia coli, in log phase growth; (2) to provide background information on motility data to study the influence of pH, viscosity, and chemotactic factors, thereby gaining a better understanding of bacterial pathogenesis. METHODS: Computer image processing technology and phase contrast microscopy (Hobson BacTracker) were used to measure several indices of bacterial motility in real time. Ten clinical isolates of each species in log phase liquid culture were studied. RESULTS: C jejuni moved fastest, with a median curvilinear velocity (CLV) of 38.76 microns/s (range 29.08 to 52.82). Next was H pylori, median CLV 25.02 microns/s (range 12.07 to 29.07). E coli was the slowest, median CLV 12.73 microns/s (range 8.20 to 18.04). The straight line velocities showed similar trends. Measurement of track linearity (TL) showed that C jejuni moved the straightest (TL 60.3%), H pylori moved in wide circles (TL 28.7%), and E coli showed spinning movement without much linear displacement (TL 18.3%). There were significant differences in these three variables between the species studied, but no significant differences in measurements of time and frequency of halts between movement runs. CONCLUSIONS: The BacTracker provides a useful technical aid for measuring several indices of bacterial motility objectively, reproducibly, and precisely, which is difficult to achieve without computer assistance. Accurate quantification of motility provides a basis for studying the factors which influence bacterial motility. It can provide phenotypic measurements of the effect of flagellar gene depletion.

Bacterial Physiological Phenomena↗
PubMed1975

Phagocytosis by muscle cells.

Electron microscopy of intact tissue and cultured smooth muscle suggested the occurrence of phagocytosis of necrotic cells by normal vascular smooth muscle cells. Phagocytosis was further studied in vitro in muscle cells from guinea pig aorta, guinea pig vas deferens, and striated muscle from chick embryo by light and electron microscopy. The uptake of yeast cells (approximately 2-mum. diameter) into living cultured smooth muscle cells from guinea pig aorta was observed with phase contrast microscopy and differential interference (Nomarski) optics. Periodic acid-Schiff staining showed the yeast cells lined up in the cytoplasm of the muscle cells. Electron microscopy confirmed the uptake of yeast cells by differentiated muscle cells. Some of the yeast in the smooth muscle cells showed signs of disintegration suggesting digestion within the phagosome. Spontaneously contracting cultured smooth, cardiac and skeletal muscle cells incubated with latex spheres (0.3-mum. diameter) showed uptake and sequestration of the spheres. The ability of muscle cells to phagocytose necrotic cells and other particulate matter may be important in atherogenesis and in the repair of tissue after injury.

Animals↗
PubMed1990

[Effect of macrophage-derived growth factor on the cell cycle kinetics of cultured arterial smooth muscle cells].

Effects of macrophage-derived growth factor on the progression of the cell cycle of cultured aortic smooth muscle cells were investigated by using rabbit peritoneal macrophage conditioned-medium (M phi-CM) as the source of the growth factors. DNA content of individual SMC in the cell population which had been exposed either to platelet-poor plasma serum (PPPS) or to M phi-CM was also measured by flow cytometry and microspectrophotometry, as well as by phase contrast microscopy. The results showed that the cells quiescent in PPPS medium had a high population not only in G0/G1 but also in G2 phase. This indicated that the cell cycle process was blocked both in G0/G1 and G2 phase. The cell cycle distribution of SMCs exposed to M phi-CM for 48 hours was very similar to that cultured in 10% fetal calf serum medium. It is suggested that M phi-CM can promote the cell growth-arrested in ppps medium passing through G0/G1 into S phase as well as through G2 into M phase.

Animals↗
PubMed2001

Role of von Willebrand factor in tumour cell-induced platelet aggregation: differential regulation by NO and prostacyclin.

1. We have studied the effects of a novel agonist, solid-phase von Willebrand Factor (sVWF), on tumour cell-induced platelet aggregation (TCIPA). 2. Washed platelet suspensions were obtained from human blood and the effects of HT-1080 human fibrosarcoma cells and sVWF on platelets were studied using aggregometry, phase-contrast microscopy, and flow cytometry. 3. Incubation of platelets with sVWF (1.2 microg ml(-1)) and HT-1080 cells (5 x 10(3) ml(-1)) resulted in a two-phased reaction characterized first by the adhesion of platelets to sVWF, then by aggregation. 4. TCIPA in the presence of sVWF was inhibited by S-nitroso-glutathione (GSNO, 100 microM) and prostacyclin (PGI(2), 30 nM). 5. Platelet activation in the presence of tumour cells and sVWF resulted in the decreased surface expression of platelet glycoprotein (GP)Ib and up-regulation of GPIIb/IIIa receptors. 6. Pre-incubation of platelets with PGI(2) (30 nM) resulted in inhibition of sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa as measured by flow cytometry using antibodies directed against both non-activated and activated receptor. In contrast, GSNO (100 microM) did not affect sVWF-tumour cell-stimulated platelet surface expression of GPIIb/IIIa. 7. Flow cytometry performed with PAC-1 antibodies that bind only to the activated GPIIb/IIIa revealed that GSNO (100 microM) caused inhibition of activation of GPIIb/IIIa. 8. The inhibitors exerted no significant effects on TCIPA-mediated changes in GPIb. 9. Thus, sVWF potentiates the platelet-aggregatory activity of HT-1080 cells and these effects appear to be mediated via up-regulation of platelet GPIIb/IIIa. 10. Prostacyclin and NO inhibit TCIPA-sVWF-mediated platelet aggregation. The mechanisms of inhibition of this aggregation by PGI(2) differ from those of NO.

Blood Platelets↗
PubMed1989

Tissue reaction after silicone and poly(methyl methacrylate) intraocular lens implantation: a light and electron microscopy study in a rabbit model.

We describe the histopathologic results of extracapsular lens extraction and silicone and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) implantation in 36 rabbit eyes. Phase-contrast microscopy was used to examine precipitates on IOL surfaces and posterior capsules. Semithin and ultrathin sections were taken from the central cornea, anterior uvea, capsular bag, and retina near the posterior pole. The follow-up was one to 16 weeks. Silicone IOLs did not cause significantly less precipitation than PMMA IOLs. Precipitates consisted of spindle-shaped fibroblast-like cells, various forms of inflammatory cells and multinucleated giant cells, single melanophages, and irregularly arranged birefringent collagen fibers. Corneal endothelial edema was slightly more prominent in PMMA IOL implanted eyes. Significant retinal edema in the posterior pole area was not observed with either of the two lens types. Severe precipitation in the form of large clusters of pigment cells and inflammatory reactions seemed to depend on mechanical trauma (iris capture and lens dislocation) and individual animal reactions, but not on the lens type used.

Animals↗
PubMed2001

Cellular overexpression of heme oxygenase-1 up-regulates p21 and confers resistance to apoptosis.

BACKGROUND: Induction of heme oxygenase-1 (HO-1) protects against diverse insults in the kidney and other tissues. We examined the effect of overexpression of HO-1 on cell growth, expression of p21, and susceptibility to apoptosis. METHODS: LLC-PK1 cells were genetically engineered to exhibit stable overexpression of HO-1. The effects of such overexpression on cell growth, the cell cycle, and the cell cycle-inhibitory protein, p21, were assessed; additionally, the susceptibility of these HO-1 overexpressing cells to apoptosis induced by three different stimuli (TNF-alpha/cycloheximide, staurosporine, or serum deprivation) was evaluated by such methods as the quantitation of caspase-3 activity, phase contrast microscopy, and the TUNEL method. RESULTS: HO-1 overexpressing LLC-PK1 cells demonstrated cellular hypertrophy, decreased hyperplastic growth, and growth arrest in the G0/G1 phase of the cell cycle. HO-1 overexpressing cells were markedly resistant to apoptosis induced by TNFalpha/cycloheximide or staurosporine as assessed by the caspase-3 activity assay. Such overexpression also conferred resistance to apoptosis induced by serum deprivation as evaluated by the TUNEL method; in these studies, inhibition of HO attenuated the resistance to apoptosis. Expression of the cyclin dependent kinase inhibitor, p21CIP1, WAF1, SDI1, as judged by Northern and Western analyses, was significantly increased in HO-1 overexpressing cells, and decreased as HO activity was inhibited. Moreover, this reduction in expression of p21 attendant upon the inhibition of HO activity in HO-1 overexpressing cells paralleled the loss of resistance of these cells to apoptosis when HO activity is inhibited. The pharmacologic inducer of HO-1, hemin, increased expression of p21 in wild-type cells and decreased apoptosis provoked by TNF-alpha/cycloheximide. CONCLUSION: Cellular overexpression of HO-1 up-regulates p21, diminishes proliferative cell growth, and confers marked resistance to apoptosis. We speculate that such up-regulation of p21 contributes to the altered pattern of cell growth and resistance to apoptosis. Our studies uncover the capacity of HO-1 to markedly influence the cell cycle in renal epithelial cells. In light of the profound importance of the cell cycle as a determinant of cell fate, we speculate that the inductive effect of HO-1 on p21 and the attendant inhibitory effect on the cell cycle provide a hitherto unsuspected mechanism underlying the cytoprotective actions of HO-1.

Animals↗
PubMed2001

Unique nucleoid structure during cell division of Thermococcus kodakaraensis KOD1.

The nucleoid structure and the partition in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 were observed by a combination of phase-contrast microscopy and fluorescence microscopy. The nucleoids occurred as rounded fluorescent foci centrally located in the cells and as differences in fluorescence intensity between exponential and stationary phases. The cellular space occupied by the nucleoid in the stationary phase was larger than that in the exponential phase. Various shapes of nucleoid in the exponential-phase cells were observed, indicating that nucleoid separation was processed under cell cycle control. The number of cells which showed distinctive division stages was counted and the proportions of dividing cells were determined. About half of the observed cells were in the replication stage. More than 40% of the counted cells possessed a fully replicated but not separated form of nucleoid. Only 8% of the total cells clearly showed visible constriction. These results suggested that the post-replication period before cell division was relatively as long as the eucaryal gap period (G2); however, the period of visible cell constriction was almost the same as that of the bacteria.

Journal Article↗
PubMed1984

Phagocytosis of hepatocyte mitochondria by rat Kupffer cells in vitro.

Kupffer cells in primary culture bind and endocytose rapidly added rat liver mitochondria. Using phase contrast microscopy various stages of the uptake and digestion of these organelles were documented. Activities of mitochondrial enzymes within the Kupffer cells increased during the early phase of phagocytosis; they later declined, reaching the endogenous level of the Kupffer cell mitochondria after 3 to 4 h. The uptake was enhanced in the presence of heparin or rat serum, while iodoacetate, cytochalasin B or anti-fibronectin antisera were inhibitory. The transient presence of enzymatically active hepatocyte mitochondria renders Kupffer cells capable of producing urea. This mechanism partially explains earlier observations of urea formation in non-parenchymal rat liver cells.

Animals↗