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PubMedDate not supplied

Establishment of vessel-like structures in long-term three-dimensional tissue culture of myocardium: an electron microscopy study.

To assess whether long-term three-dimensional (3D) tissue culture of myocardium enables the in vitro establishment of vessel-like structures, myocardial tissue from newborn mice was incubated under conditions of 3D culture for at least 3 weeks, and studied by phase-contrast microscopy, conventional histology, immunohistochemistry, and electron microscopy. During 3 weeks of culture, a mean 24.35 +/- 3.74% of all aggregates contracted spontaneously. The contracting aggregates displayed a tissue-like architecture with small basal and apical zones, and a large central zone. The basal and apical zone consisted of immature mesenchymal cells. The underlying shell of the aggregate contained many cardiomyocytes. Vessel-like structures were found concentrated within the aggregates. Immunohistochemistry showed that up to 15% of the cells in the central zone of the aggregate were positive for the endothelial-specific BS-I lectin. Vessel-like structures were formed by cells, which often showed intracytoplasmatic lumena. Surrounding the neocapillaries, structures of a rudimentary basal membrane could be detected. A 3D culture of myocardial tissue permits the establishment of a rudimentary capillary network within the tissue aggregates, which presumably guarantees a sufficient tissue perfusion up to a maximum aggregate diameter of approximately 900 microm.

Adaptation, Physiological↗
PubMedDate not supplied

Influence of wet fixation, staining techniques, and storage time on bull sperm morphology.

The influence on sperm morphology of different methods for preparation of semen and of storage in a fixative solution was examined in 27 beef bulls subjected to a regular breeding health examination. Sperm head morphology under light microscopy did not differ between smears of fresh semen stained with carbol-fuchsin-eosin (Williams staining) or Nigrosin-Eosin. Nor was there any difference between samples stained immediately after collection and those stained after 1 month of storage at + 4 degrees C in buffered formal-saline solution. Formol-saline fixed spermatozoa examined in wet preparations under phase contrast microscopy had a higher prevalence of acrosome defects and cytoplasmic droplets than stained smears of fresh semen under light microscopy. One month of storage in formol-saline did not affect the prevalence of acrosome defects or cytoplasmic droplets. There was no influence of fixation method (wet or dry), staining, examination technique, or storage time on midpiece or sperm tail morphology. The affinity of spermatozoa to eosin at staining with Nigrosin-Eosin ("live and dead count") did not differ between fresh semen and spermatozoa that had been stored in formol-saline for 1 month. It is concluded that bull semen can be stored for at least 1 month at + 4 degrees C in buffered formal-saline without major changes in sperm morphology. Furthermore, examination of wet preparations of fixed spermatozoa under phase contrast microscope is likely to yield the most accurate results for morphological characteristics like acrosome morphology and cytoplasmic droplets.

Animals↗
PubMed1986

Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.

DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs. Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes. Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy. Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed. About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin. ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes. However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion.

Adenosine Triphosphate↗
PubMed1978

Intracellular fibres in cultured cells: analysis by scanning and transmission electron microscopy and by SDS-polyacrylamide gel electrophoresis.

The 3-dimensional structure of the fibrous cytoskeleton of 3T3 cells was examined by scanning electron microscopy of cells extracted with the non-ionic detergent Triton X-100. Detergent-extracted cells consist of the nucleus and an extensive system of fibres, the largest of which correspond to stress fibres visible by phase-contrast microscopy. The system of fibres, which is coterminous with the borders of the native cell, remains firmly adherent to the substratum. The major fibres branch into smaller fibrils which appear to end by ravelling out into fine filaments that constitute a matted network in a plane very close to that of the substratum. In the nuclear region all the major fibres pass over the top of the nucleus, where they may also branch into a system of fine fibrils. Thin-section transmission electron microscopy in conjunction with heavy meromyosin treatment of extracted cells shows the fibres to be composed of native F-actin. Intermediate filaments are also present, and are prominent in the matted network, together with actin filaments. The major proteins of the residue are identified by SDS-polyacrylamide gel electrophoresis as actin, a 56000 Dalton peptide, and histones. Also present are myosin heavy chain, peptides of 225,000 and 250,000, and minor bands at 60,000 and 94,000 Daltons. The non-ionic detergent extracts 70% of the cellular protein, including 50% of the actin and 75% of the myosin. The Triton-insoluble fraction of 3T3 cells appears to constitute, in addition to the nucleus, a stable cytoskeletal system, composed largely of contractile proteins and 10-nm filaments, which functions in maintenance of cell shape, in substratum adhesion, and in positioning the nucleus within the cell.

Cell Adhesion↗
PubMed1997

Microtubules are involved in bafilomycin A1-induced tubulation and Rab5-dependent vacuolation of early endosomes.

The small GTPase Rab5 is an important regulator of membrane fusion in the early endocytic pathway. Here we have studied at the light microscopy level the morphology of early endosomes in MDCK cells stably expressing a GTPase-deficient Rab5 mutant, Rab5 Q79L, N-terminally tagged with a myc-epitope. These cells contain large vacuoles, readily visible by phase-contrast microscopy. Confocal immunofluorescence microscopy showed the presence of the epitopetagged protein on large perinuclear vacuoles, as well as on smaller peripheral structures. A subset of the perinuclear vacuoles appeared to colocalize with the late endosomal GTPase, Rab7. In addition, a population of very large Rab7-positive, Rab5 Q79L-negative structures were observed, suggesting that an increase in the size of early endosomes may be accompanied by an increased size of later or more mature endocytic structures. Using antibodies against the myc epitope and the early endosomal autoantigen EEA1 as markers, we found that endosomes in wild-type and mutant MDCK cells rapidly tubulate in the presence of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase. Elongated or tubular endosomes partially colocalized with microtubules and were redistributed upon preincubation with the microtubule depolymerizing agent nocodazole before bafilomycin A1 treatment. Treatment of the Rab5 Q79L expressing cells with nocodazole alone led to a spatial redistribution and a significant decrease in the size of EEA1-positive structures, whereas their number increased. These results implicate microtubules in the bafilomycin A1-induced tubulation of endosomes as well as in the vacuolation of endosomes caused by Rab5 Q79L.

Animals↗
PubMed1992

Isolation and biochemical characterization of the human sperm tail fibrous sheath.

The isolation and biochemical characterization of the human sperm tail fibrous sheath (FS) is described for the first time. Initially, the solubilization properties of the FS were assessed immunocytochemically using GDA-J/F3 and RT97 monoclonal antibodies (MoAbs) and morphologically by electron microscopy. Following extensive investigations to optimize the conditions for the FS isolation, a simple method was developed which involved sequential extraction of the flageller components with Triton-dithiothreitol (DTT) and urea-DTT. The procedure was monitored by phase contrast microscopy and the purity of the FS preparations was confirmed by electron microscopy. SDS-PAGE of the isolated FS revealed seven major protein bands with mol. wt of 97, 76, 62, 55, 33, 28 and 25 kDa. In Western blotting, the reaction of RT97 MoAb with supernatants from the various extraction steps and the isolated FS indicated that its target antigen (AJ-p97) was an integral FS product and that disulphide bonding was probably involved in its stabilization. The reactivity of normal and aprotruded sperm tails with GDA-J/F3 and RT97 MoAbs was not affected by Triton while the GDA-J/F3 staining of the cytoplasmic matrix of other abnormal spermatids was abolished, thus suggesting variation in the biochemical properties of GDA-J/F3 in normal and abnormal germ cells. These and other data indicate that the FS could be a modified form of intermediate filament.

Antibodies, Monoclonal↗
PubMed1995

Stability of parenteral nutrition admixtures containing organic phosphates.

The use of organic phosphates to avoid calcium phosphate precipitation in parenteral nutrition mixtures has been proposed. The purpose of this study was to evaluate the stability of total parenteral nutrition admixtures containing glucose-1-phosphate or glycerol phosphate as the phosphate source over 3 days. Three parenteral nutrition admixtures, each containing glucose-1-phosphate (30.0 mmol), glycerol phosphate (31.4 mmol) or inorganic phosphate (30.0 mmol), and their corresponding aqueous phases were prepared in 3-L ethylene vinyl acetate plastic bags and infusion bottles, and stored at 5 +/- 1 degrees C or 22 +/- 3 degrees C without light protection. Physical stability analysis and sampling for chemical analysis was performed at 0, 24, 48 and 72 h. Aqueous phases were subjected to physical stability analysis, including pH measurement, visual inspection and nephelometry. Admixtures were subjected to physical stability analysis consisting of pH measurement, and evaluation of emulsion stability by visual inspection, degree of creaming, phase contrast microscopy, zone sensing technique and photon correlation spectroscopy. Chemical analyses of amino-acids, dextrose, triglycerides, phospholipids, Na, K, Cl, Mg, Ca, glucose-1-phosphate, glycerolphosphate and inorganic phosphate were performed. No precipitation was detected in any of the aqueous phases. Admixtures remained acceptable with respect to visual and microscopic appearance, mean droplet diameter and droplet size distribution. All nutrient concentrations assayed in the three admixtures remained constant over the study period. Total parenteral nutrition admixtures for adult patients containing glucose-1-phosphate or glycerolphosphate are physically and chemically stable for 3 days when stored under refrigeration or controlled room temperature without light protection.

Journal Article↗
PubMed2000

Coexistent adenoviral keratoconjunctivitis and Acanthamoeba keratitis.

A 17-year-old youth presented with bilateral follicular conjunctivitis and nummular subepithelial corneal infiltrates. Failure of this to settle in an outpatient setting led to corneal scraping with microscopy and culturing for bacteria, fungi, Herpes simplex, adenovirus and Acanthamoeba as an inpatient. Polymerase chain reaction analysis of corneal cells was positive for adenovirus, and culture on live Escherichia coli-coated agar plates was positive for Acanthamoeba by phase contrast microscopy on day two. We conclude that Acanthomoeba infection can complicate adenoviral keratoconjunctivitis. This observation is in keeping with previously reported modes of infection by Acanthamoeba, whereby any epithelial breach seems to allow inoculation of the eye by this opportunistic organism.

Acanthamoeba↗
PubMed1988

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture.

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.

Animals↗
PubMed1998

Development of a method for the determination of low contents of asbestos fibres in bulk material.

Asbestos is a category 1 carcinogen under the EU classification, but in the absence of a method to quantify asbestos in a matrix at the 0.1% level, there has been a delay in implementing relevant directives to asbestos. An analytical scheme for identification and quantification of asbestos using polarised light microscopy (PLM) and phase contrast optical microscopy (PCM) has now been developed. When used on artificial mixtures by an experienced laboratory, it achieved the required target performance, at 0.1% asbestos concentration by mass in a bulk sample, to obtain a result, which with 90% probability, is correct within a factor of two. The method of identification by PLM and quantification by PCM has been assessed by interlaboratory comparisons. The method begins with an initial identification using PLM, and depending on asbestos type and matrix a combination of preparation procedures are used to produce the analytical filter. A gentle comminution method was used which reduces the risk of overmilling. The asbestos mass percentage on the filter is quantified using PCM in combination with a PLM attachment for identification of possible non-asbestos fibres. The final method is supported by efficient methods for fibre identification for size determination and calculation of total fibre volume. A statistical analysis of mass concentration estimates was made and the effect of preferred orientation of fibres on the analytical filter was quantified.

Asbestos↗
PubMed1989

NBD-taurine uptake by alpha-type carbonic anhydrase cells of turtle bladder.

The uptake of the fluorescent anion, N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)aminoethanosulfonic acid-taurine (NBD-T) was examined in sheets of turtle bladder epithelial cells after either serosal or mucosal exposure to the fluorescent anion. Serosal uptake of NBD-T into a population of epithelial cells was demonstrated in thin epithelial sheets scraped from bladder sacs exposed to low (0.5 mM) concentrations of NBD-T sufficient to cause fluorescence in turtle erythrocytes. In nine sheets NBD-T fluorescence was observed in 14.4 +/- 1.3% of epithelial cells; by phase-contrast microscopy of the same fields, these cells were nongranular or carbonic anhydrase (CA) cells. An additional 8.1 +/- 1.0% of cells was nongranular, but failed to exhibit fluorescence after serosal NBD-T exposure, consistent with a beta-CA cell population. Apical uptake of NBD-T into epithelial cells was demonstrable only at high (5 mM) mucosal concentrations in both thin sheets and superfused bladder segments. In thin sheets, NBD-T fluorescence was observed in 15.8 +/- 0.7% of cells and appeared to have a punctate subapical distribution; phase-contrast of the same fields (n = 46) revealed a nongranular cell population of 21.9 +/- 0.9%. Nongranular cells corresponded closely to CA cells as identified with carboxyfluorescein fluorescence. Hence, apical NBD-T was taken up primarily by alpha-CA cells. In conclusion, NBD-T is transported by alpha-CA cells either across the basolateral membrane with high affinity comparable to that of turtle red cell band 3 protein or across the apical membrane with low affinity via a less-specific mechanism such as endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

In vitro spheroplast and L-form induction within the pathogenic nocardiae.

Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.

Culture Media↗
PubMed2002

TGF-beta isoform release and activation during in vitro bronchial epithelial wound repair.

Restitution of an epithelial layer after environmental or biological damage is important to maintain the normal function of the respiratory tract. We have investigated the role of transforming growth factor (TGF)-beta isoforms in the repair of layers of 16HBE 14o(-) bronchial epithelial-derived cells after damage by multiple scoring. ELISA showed that both latent TGF-beta1 and TGF-beta2 were converted to their active forms 2 h after wounding. Time-lapse microscopy showed that the addition of TGF-beta1, but not TGF-beta2, progressively increased the rate of migration of damaged monolayers at concentrations down to 250 pg/ml. This increase was blocked by addition of a neutralizing TGF-beta1 antibody. Phase-contrast microscopy and inhibition of proliferation with mitomycin C showed that proliferation was not required for migration. These results demonstrate that conversion of latent to active TGF-beta1 and TGF-beta2 during in vitro epithelial wound repair occurs quickly and that TGF-beta1 speeds epithelial repair. A faster repair may be advantageous in preventing access of environmental agents to the internal milieu of the lung although the production of active TGF-beta molecules may augment subepithelial fibrosis.

Antibodies, Monoclonal↗
PubMed2001

Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells.

Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1-encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin-Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F-actin in tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin-stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild-type and sipA mutant strains of S. typhimurium. Time-lapse phase-contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild-type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild-type bacteria or failed to invade at all. Furthermore, although wild-type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild-type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.

Actins↗
PubMed1991

[The emissions of fiber particles into the atmosphere in the region of an asbestos-processing industry].

Examination is carried out on dust taken from the atmospheric air in the town of Sevlievo, where a plant for asbestos products is situated. The concentrations of fibre particles are determined by the methods of light and electron microscopy. The results are compared with the emissions of fibres in the air of a control town. There is a significant contribution of an industrial source of emission in the atmospheric pollution by fibre particles. It was proved that the method of light-phase contrast microscopy is not suitable for analyses of low concentrations of fibres in the air. As only reliable method is that of electron microscopy.

Air Pollutants↗
PubMed1995

Establishment and characterization of a new human lung poorly differentiated adenocarcinoma cell line, GLL-1, producing carcinoembryonic antigen (CEA) and CA19-9.

A new human lung poorly differentiated adenocarcinoma cell line producing carcinoembryonic antigen (CEA) and CA19-9, designated GLL-1, was established from a serial xenograft established in our laboratory. In this paper, we investigated the characteristics of GLL-1 cells. The cell line grew in a monolayer and had a polygonal epithelioid appearance in phase-contrast microscopy. The doubling time of GLL-1 cells during exponential growth was approximately 28 h in RPMI-1640 with 10% fetal calf serum. Positive reactions for CEA and for CA19-9 were seen within the cytoplasm of GLL-1 cells by immunohistochemical staining. In log phase, the fractions for G0/G1, S and G2/M were 76.2%, 20.0% and 3.8%, respectively. Chromosome analysis revealed that GLL-1 cells contained the modal number of 72 chromosomes (range 66-97). GLL-1 cells exhibited numerous numerical and structural abnormalities. The Do value for radiation sensitivity, calculated by taking the reciprocals of the slope of the radiation dose response curve, was 109 cGy. The extrapolation number (n) for the radiation response curve was 1.57. Tumor take was observed in nude mice inoculated subcutaneously in the back with 2 x 10(6) GLL-1 cells. The histopathological features of the tumor induced by inoculation of GLL-1 cells were also the same as the original lung cancer.

Adenocarcinoma↗
PubMed1987

Ingestion of Giardia lamblia trophozoites by human mononuclear phagocytes.

Mononuclear phagocytes may be important effector cells against Giardia lamblia. Human monocyte-derived macrophages were incubated with G. lamblia trophozoites in 13% heat-inactivated autologous serum. At a G. lamblia/macrophage ratio of 1:1, the number of trophozoites ingested per 100 macrophages ranged from 1 to 12 at 0.5 h and increased for all donors (n = 6) to 10 to 92 at 8 h. Ingestion was confirmed by electron microscopy. Increasing the parasite/phagocyte ratio to 5:1 increased the percentage of macrophages with adherent but not ingested trophozoites. Incubating Giardia cells and macrophages with 20% immune serum increased ingestion of parasites eightfold, indicating that anti-G. lamblia antibody can enhance ingestion. Both phase-contrast microscopy and electron microscopy documented trophozoite destruction within macrophages. Ingestion of parasites elicited an oxidative burst as measured by luminol-enhanced chemiluminescence. In vitro, Giardia trophozoites were killed by greater than or equal to 5 X 10(-5) M H2O2. Fusion of lysosomes with parasite-containing phagosomes was suggested by acridine orange-stained preparations. Human macrophages have the capacity to ingest Giardia trophozoites and to kill intracellular parasites, possibly by oxidative microbicidal mechanisms.

Animals↗
PubMed2002

Evaluation of an automated urinalysis system for testing urine chemistry, microscopy and culture.

AIMS: This study was designed to evaluate the performance of an automated urinalysis system that utilised three commercially available instruments, the Clinitek Atlas, Sysmex UF-100 and the Alifax Uro-Quick. METHODS: The results of the automated system for 818 urine samples were compared with the results of manual processing which consisted of phase contrast microscopy, manual dipstick chemistry analysis and culture onto solid media. RESULTS: The correlation between the two methods for urine chemistry was excellent with a concordance of 89, 97, 100 and 98% for pH, blood, glucose and protein, respectively. The quantification of red blood cells and white blood cells had an R2 of 0.855 and 0.92, respectively. A difference scatter plot indicated a trend towards the manual cell count being greater than the UF-100 count as both the red and white blood cell count increased. There was 98% agreement between the automated process and manual culture. CONCLUSIONS: The automated urinalysis system is fully integrated and allows for the cross-checking of urine chemistry and microscopy as well as electronic transfer of data. The automated process was used as a screening procedure and some manual testing was necessary. Automation of urinalysis offers a reduction in variation and has comparable results to manual testing.

Autoanalysis↗