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PubMed2005

Histone deacetylase inhibitors differentially mediate apoptosis in prostate cancer cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have shown significant anti-proliferative and apoptotic properties on various cancer cells, including prostate cancer, and are therefore being evaluated as treatment modalities. However, the specific effect of HDAC inhibitors on androgen-sensitive and androgen-independent cell lines have not been thoroughly studied which we hypothesized could be different. We therefore assessed whether three structurally unrelated HDAC inhibitors, trichostatin A (TSA), depsipeptide (FR901228), and sodium butyrate, affect cell death in the prostate cancer cell lines LNCaP, DU-145, and PC-3. METHODS: To investigate the extent and the nature of cell death, we used Trypan blue exclusion assay, phase-contrast light microscopy, fluorescence microscopy, and Western blot analyses. RESULTS: At concentrations where they potentiate transcriptional activation, all three HDAC inhibitors induced cell death in LNCaP and DU-145 cells, but not in PC-3 cells, within the timeline of the experiments. HDAC inhibitor-induced cell death in LNCaP and DU-145 cells showed several characteristic apoptotic features, such as cell shrinkage, nuclear condensation, and poly(ADP) ribose polymerase cleavage. However, there were differences in the way LNCaP and DU-145 cells responded to treatment with various HDAC inhibitors. For example, whereas TSA and FR901228 were more effective in inducing apoptosis in LNCaP cells compared with DU-145 cells, the reverse was true for sodium butyrate. Moreover, within the same cell line, TSA, FR901228, and sodium butyrate exhibited different potencies for induction of apoptosis. CONCLUSIONS: Collectively, these results suggest that the response of prostate cancer cells to HDAC inhibitors is not uniform, but cell line and inhibitor specific. Given that prostate cancer is generally a multiclonal disease representing different cell lineages, it is important to develop HDAC inhibitors that will be effective against all of these cell types.

Apoptosis↗

PubMed1989

Kinetics of C-reactive protein in acute viral hepatitis.

The significance of C-reactive protein (CRP) in acute viral hepatitis was studied by measuring the serum CRP level in patients with acute hepatitis type A (AHA), B (AHB), and non-A, non-B (AHNANB) and examining its localization in liver biopsy specimens by the immunohistochemical method. The mean value of the serum CRP level determined by enzyme immunoassay (EIA), was markedly increased in the acute phase of AHA and AHB, particularly the former. It decreased rapidly in both AHA and AHB during the convalescent phase, but was generally low in AHNANB with no marked difference between the acute phase and the convalescent phase. Under light microscopy, CRP was stained in the cytoplasm of hepatocytes, and immuno-reactive products were observed in the rough endoplasmic reticulum (RER) by electron microscopy. In the acute phase, the intensity of staining was slightly greater in AHA, decreasing during the convalescent phase in AHA and AHB, but only weak staining was observed in all patients with AHNANB. Evaluation of CRP may be useful for clarification of differences in clinical manifestations and the mechanisms of inflammation among different types of hepatitis.

Acute Disease↗

PubMed1984

Human ciliary epithelia in monolayer culture.

The ciliary epithelia of human (one to 12 months old) ciliary processes were isolated by trypsin and EDTA, cultured in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal calf serum and examined by phase and electron microscopy. The primary cultures were maintained for three to four months. Only a few non-pigmented epithelia adhered and none of them proliferated. After the first passage the culture seemed to consist of only the pigmented epithelia. Most cells were densely pigmented at first, then became less pigmented during successive proliferations. Half of the cells remained densely pigmented after the first subculture, another half remained less pigmented. The cells started to lose their pigment granules at four to six weeks in culture. After three months of culture, the cell sheets became entirely unpigmented. In thin section, most of the pigment granules in the cells at two weeks in culture were pre-melanosomes, and half of them were at the earliest stage of pre-melanosomes. Monolayer cells possessed basement membranes. At 14 weeks in culture, most cells established an apparent polarity, contained well-developed Golgi apparatus and rough endoplasmic reticulum and intermediate filaments, but no pigment granules. A bundle of intermediate filaments was found in the perinuclear cytoplasm. Multilayer cells presented a typical apex-to-apex and base-to-base configuration , and the extracellular material was detected only in the base-to-base intercellular spaces. Our culture system provided differentiated cells derived from the pigment epithelia of human ciliary processes.

Cells, Cultured↗

PubMed1993

Sites of plasmalemmal expansion in growth cones.

Studies on plasmalemmal expansion in isolated nerve growth cones identified large, clear vesicles characteristically found in growth cones as the plasmalemmal precursor. The present article examines these plasmalemmal precursor vesicles (PPVs) in greater detail in the intact cell. (a) Pulse-chase experiments with the phospholipid precursor, [3H]glycerol, followed by radioautographic analysis show that PPVs in distal neurites and growth cones are labeled prior to equilibration of the label with the plasmalemma. (b) Pulse-chase experiments with lectin-ferritin conjugates demonstrate that PPVs are not endocytotic, that they contain lectin receptors, and that, during growth, patches of lectin receptors appear on the plasmalemma covering PPV clusters. (c) Freeze-fracture studies show that this plasmalemma shares with PPVs a paucity of intramembrane particles. (d) Lectin labeling experiments and freeze-fracture analysis demonstrate, furthermore, that the plasmalemma forms a network of invaginations at the base of PPV clusters. (e) Correlative studies indicate that the refractive 'vacuoles' seen in growth cones by phase-contrast light microscopy correspond to the PPV clusters seen at the ultrastructural level. These results confirm the identity of the plasmalemmal precursor in the intact cell and demonstrate that PPV clusters form distinct, dynamic organelles specialized for plasmalemmal expansion in the growth cone.

Animals↗

PubMed1990

Fiber exposure reassessed with the new indices.

The concentration of airborne fibers longer than 5 microns, thinner than 3 microns, and with an aspect ratio exceeding 3 as counted by phase contrast optical microscopy is the most widely used fiber exposure index. Recently, more adequate, specific exposure indices for asbestosis, lung cancer, and mesothelioma risk have been suggested by Lippmann (1988, Environ. Res., 46, 86-106). The consequences of using these indices are examined on the basis of calculations for a broad range of theoretical and published size distributions. Optical microscopy appears to be a good predictor of the exposure indices for asbestosis and for lung cancer after scaling. Only fibers longer than about 3 microns need to be counted in a transmission electron microscope. The lung cancer index still cannot explain the large differences of risk among chrysotile exposures. Both the mesothelioma exposure index and the ratio mesothelioma to lung cancer index ranks in order of increasing risk: wollastonite, glass and mineral wool, amosite, glass microfibers, chrysotile, and crocidolite. Amosite is thus not ranked according to epidemiological evidence. Detailed size information should be made available so that the size criteria can be adjusted. It may still prove necessary to use fiber type specific concentration limits.

Asbestos↗

PubMed1990

Influence of chondroitin sulfate, heparin sulfate, and citrate on Proteus mirabilis-induced struvite crystallization in vitro.

Struvite crystals were produced by Proteus mirabilis growth in artificial urine, in the presence of a number of naturally occurring crystallization inhibitors. The use of phase contrast light microscopy enabled the effects of added chondroitin sulfate A, chondroitin sulfate C, heparin sulfate, or sodium citrate, on struvite crystal growth rates to be rapidly monitored as changes in crystal habit. Struvite crystals formed as a consequence of the urease activity of P. mirabilis under all chemical conditions. In the absence of inhibitor, early crystal development was marked by large quantities of amorphous precipitate, followed immediately by the appearance of rapidly growing X-shaped or planar crystals. Addition of the glycosaminoglycans, chondroitin sulfate A, chondroitin sulfate C, or heparin sulfate to the artificial urine mixture had no effect on the rate of crystal growth or appearance. When sodium citrate was present in elevated concentrations, crystal appearance was generally slowed, and the crystals assumed an octahedral, slow growing appearance. None of the added compounds had any influence on bacterial viability, pH, or urease activity. It is therefore likely that the inhibitory activity displayed by sodium citrate might be related to its ability to complex magnesium or to interfere with the crystal structure during struvite formation. From these experiments it would appear that citrate may be a factor in the natural resistance of whole urine to struvite crystallization.

Chondroitin Sulfates↗

PubMedDate not supplied

Glycerol derivatives of cutin and suberin monomers: synthesis and self-assembly.

Glycerol derivatives of cutin and suberin monomers were synthesized by acid catalysis. Their dispersion in an aqueous solution was examined by phase contrast microscopy, neutron scattering, and solid state NMR. It is shown that the phase behavior strongly depends on the nature of the derivatives forming either lumps of aggregated membranes or well dispersed membranes.

Glycerol↗

PubMed2000

Nonhomologous bioinjectable materials in urology: 'size matters'?

OBJECTIVE: To assess the factors that influence how particles might become fixed in tissues or migrate from them, by measuring the size of the injectable particles, their susceptibility to phagocytosis and their affinity for fibroblast attachment in culture. MATERIALS AND METHODS: The particle size of three types of particulate unphysiological bioinjectable material, i.e. Urocol (Genesis Medical, Ltd., London), Macroplastiquetrade mark (Uroplasty Ltd., Reading, UK) and Urethrin (Mentor Medical Systems, Wantage, UK) was analysed using phase-contrast light microscopy and confocal microscopy. Human monocytes from peripheral blood were incubated with the three materials in phagocytic studies, where ingestion was determined by confocal microscopy. A fibroblast cell line was used to ascertain the ability of the particles to act as a substrate for cell attachment in culture. RESULTS: The mean (SEM) maximum particle diameters of Macroplastique, Urethrin and Urocol were 209 (5.10) microm, 49 (1.52) microm and 14 (0.39) microm, respectively. Rat peritoneal macrophages and human peripheral blood monocytes commonly ingested Urocol particles; the phagocytosis of Urethrin was rare and that of Macroplastique was not detected. Fibroblasts adhered to Urocol paste and Urethrin particles, but not to Macroplastique. CONCLUSION: Published reports of particle size and phagocytosis are confusing, but a relationship clearly exists. Macroplastique is the largest particle and is least likely to be phagocytosed by human mononuclear phagocytes. Urocol paste is the slowest to dissipate in culture conditions; the flat surfaces of Urethrin, but not Macroplastique, can serve as a substrate for fibroblast anchorage.

Animals↗

PubMed1994

Sexual dimorphism and developmental change of the salivary glands in adult Culicoides variipennis (Diptera: Ceratopogonidae).

Salivary glands of adult male and female Culicoides variipennis (Coquillett) were sexually dimorphic when examined by phase contrast light microscopy. Female salivary glands were larger and more complex than those in males. Each female gland consisted of a main gland, which was subdivided into a proximal neck and a distal body with reference to the salivary duct, and four accessory glands. Each male salivary gland consisted of a pear-shaped body with a constriction, or neck, that divided it into a proximal and a distal portion, with reference to the salivary duct. Salivary glands of both sexes increased in length from emergence to day 3, followed by a sex-specific pattern of decrease. Based on these morphological observations, we suggest that the salivary glands of female C. variipennis are specialized in the production of secretory materials for blood-feeding.

Aging↗

PubMed2003

Pathways of DNA double-strand break repair during the mammalian cell cycle.

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.

Animals↗

PubMed1975

A morphological study of anaerobic bacteria from the hypolimnia of two Michigan lakes.

Dense populations of anaerobic bacteria were found sequentially layered below the thermocline in two eutrophic lakes in southwest Michigan. Phase and electron microscopy of whole cells and thin sections were used to reveal the in situ morphology of the dominant members of the community. The predominant chlorophyll-containing bacteria were identified on the basis of their morphology to be members of the genera Pelodictyon, Prosthecochloris, Clathrochloris, Chlorochromatium, Pelochromatium, Thiopedia, Thiocystis, Thiospirillum, and Chromatium. The natural morphology of these organisms is described and compared with the morphology of reported isolates, the morphology of unisolated genera was compared with previous descriptions of natural samples. Most of the organisms near the sediment-water interface and two from the upper hypolimnion have not been previously described. They have been divided into six distinct groups based on morphology; the morphological features of each group are presented. This approach, based on the morphological uniqueness of the procaryotes present, provides a satisfactory method for grouping members of the hypolimnetic community for ecological studies.

Anaerobiosis↗

PubMed1991

Are Ranvier's nodes artifacts by preparation?

To avoid artifacts, native nerve fibers were investigated by phase contrast light microscopy. It was shown that Ranvier's nodes cannot be seen. At the sites at which they can be produced by thermal and chemical effects, a tight joint of the tube ends of the 'internodes' inverted to the inside are to be found. Some structures of Ranvier's nodes do not exist in intact living nerves. This fact can also be proved by electron micrography.

Animals↗

PubMed1998

Assessment of environmental asbestos exposure in Turkey by bronchoalveolar lavage.

Environmental or domestic exposure to asbestos fibers originating from local soil is responsible for a high incidence of diseases in large rural areas of Turkey. Bronchoalveolar lavage fluids (BALF) were obtained for 65 Turkish subjects originating from these areas and for 42 Turkish controls. Asbestos bodies (ABs) and uncovered fibers (UFs) were quantified by phase contrast light microscopy. Total fiber burden was determined by transmission electron microscopy. The main asbestos types disclosed were tremolite and to a lesser extent chrysotile. AB and fiber concentrations were higher in environmentally exposed subjects (geometric mean [geometric standard deviation]: 5.20 [6.22] AB/ml, 444 [11.6] tremolite fibers/ml) than in control subjects (0.22 [1.45] AB/ml, 12.0 [15.4] tremolite fibers/ml) (p < 0.001). In subjects environmentally exposed in Turkey, AB burdens on tremolite were in the same range as those on commercial amphiboles in subjects occupationally exposed in Belgium. In Turkish subjects, values above either 1 AB/ml, 3 uncovered fiber/ml in light microscopy, or 300 fibers/ml in electron microscopy indicated usually an abnormal alveolar retention reflecting a significant cumulative exposure from environmental or domestic origin. These observations are probably valid for other areas in the world where diseases associated with environmental exposure to soil- derived asbestos fibers occur and for immigrants originating from these areas.

Adult↗

PubMed2003

Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia.

PURPOSE: Proinflammatory signaling mechanisms are implicated in the induction of retinal neovascularization (NV) during ischemic retinopathies. This study examined transcription factor (TF) AP-1, HIF-1, and NF-kappaB DNA-binding in relation to cyclooxygenase (COX)-2 and VEGF RNA and protein levels in hypoxia-triggered monkey choroidal retinal (RF/6A) endothelial cells. Effects of the carboxamide CGP43182 were tested on COX-2 and VEGF activation and prostaglandin (PG)E(2) release. METHODS: RF/6A cells were subjected to hypoxia for 1 and 3 hours, at which times RNA and proteins were isolated. Potential AP-1, hypoxia-inducible factor (HIF)-1 and NF-kappaB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE(2) levels were quantified by RT-PCR, Western analysis, and enzyme immunoassay, respectively. Tubular morphogenesis was analyzed with phase-contrast imaging microscopy. RESULTS: Nuclear AP-1, HIF-1 and NF-kappaB promoter DNA binding increased 1.5-, 4-, and 3-fold, respectively, after 1 hour of hypoxia. COX-2 RNA was elevated five- and fourfold after 1 and 3 hours of hypoxia, respectively. VEGF RNA and protein abundance lagged behind COX-2 induction but were each increased two- to threefold 3 hours after hypoxia. CGP43182 was found to inhibit NF-kappaB DNA binding, COX-2 and VEGF gene expression, PGE(2) release, and hypoxia-induced tubular morphogenesis. CONCLUSIONS: Maximum HIF-1 and NF-kappaB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1beta emulated AP-1, HIF-1, and NF-kappaB DNA binding during hypoxia and may be a novel cytokine trigger for NV. CGP43182 appears to be an effective inhibitor of NV. VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 directly and indirectly through NF-kappaB-mediated COX-2 expression and PGE(2) production.

Animals↗

PubMed2006

Cyclosporine-loaded microspheres for treatment of uveitis: in vitro characterization and in vivo pharmacokinetic study.

PURPOSE: A sustained intraocular level of immunosuppressive drug is desirable for the treatment of uveitis and other intraocular immune disorders. The objective of the present investigation was to assess the suitability of cyclosporine-loaded poly(lactic-co-glycolic acid) microspheres (CyS-PLGA-MS) to achieve this goal. METHODS: A solvent-evaporation method was used in the preparation of CyS-PLGA-MS. These microspheres were characterized for drug loading, entrapment efficiency, and in vitro release by high-performance liquid chromatography, particle size by phase-contrast light microscopy and surface morphology by scanning electron microscopy. The 3H-CyS-PLGA-MS suspension was injected into the vitreous body of healthy rabbits, and the concentration of cyclosporine in various ocular tissues and blood at predetermined intervals was measured by a scintillation counting technique and the pharmacokinetic parameters were calculated. Intravitreous administration of 3H-CyS solution was conducted as the control. RESULTS: The CyS-PLGA-MS was produced, with drug-loading ranging from 11% to 16% and a high entrapment efficiency from 86% to 98%. Microspheres were discrete, spherical particles with a diameter of approximately 50 microm. The CyS was constantly and slowly released from microspheres in the in vitro release experiment. Compared with CyS solution, microspheres prolonged the release of CyS and maintained therapeutic CyS concentrations for at least 65 days in disease-related tissues such as the choroid-retina and iris-ciliary body. The percentage of CyS released in vitro correlated well with the CyS distribution rate in vivo. CONCLUSIONS: CyS-PLGA-MS, displaying sustained intraocular release of CyS and showing advantages over CyS solution, may meet clinical needs more efficiently.

Animals↗

PubMedDate not supplied

Compound action-potential tuning curves in normal and acoustically traumatized cats.

Compound action-potential tuning curves, using a forward-masking paradigm, were developed on both a control group and a group of acoustically traumatized cats. Differences observed between the two populations included a decrease in the sharpness of the tip, in the sensitivity of the tip, and/or in the sensitivity of the tail region. Phase-contrast light microscopy was performed on all exposed ears using a celloidin-embedding technique with horizontal sectioning. Whenever an abnormality in an action-potential tuning curve was seen, histological evidence of damage to the organ of Corti in an appropriate region corresponding to the signal frequency was observed. However, several cases of damage to the cochlea were observed with normal tuning curves. Whenever the tip region of the tuning curve was elevated, evidence of damage to all three rows of outer hair cells and to the inner hair cells was seen.

Animals↗

PubMed2001

Morphological changes and recovery process in the tenotomized soleus muscles of the rat.

Tenotomized soleus muscles of adult rats were analyzed morphologically and biochemically with special reference to the recovery process. Light microscopic observations of semi-thin sections showed that the characteristic central core lesion was most extensive at 1 week after tenotomy and began to diminish in extent at 2 weeks until no trace of lesion could be seen by 6th week, as confirmed by thin-section electron microscopy. Three phases of changes in the cross-sectional area of muscle fibers after tenotomy were demonstrated by morphometry: phase I designated as the initial increase up to the 3rd day, phase II as the progressive decrease until the 4th week, and phase III as the recovery to normal or even hypertrophy. In electron microscopy, the earliest alteration of myofibrils was recognized at 3 days after tenotomy. The Z discs showed a wavy or zigzag profile with frequent longitudinal splitting of myofibrils. From the 2nd week on, muscle fibers underwent a process of recovery, replacing the central core lesion with new myofibrils in which a reassembly of thick filaments into bundles of thin filaments took place, with Z discs being aligned adjacent to the peripheral complete myofibrils. In SDS-polyacrylamide gel electrophoresis, the molar ratio of myosin to actin diminished markedly as the central core lesion developed and gradually returned to normal with time, correlating well with the loss and subsequent reassembly of thick filaments.

Actins↗

PubMed2005

A large-scale automated method for hepatocyte isolation: effects on proliferation in culture.

Large-scale sterile methods for isolating hepatocytes are desirable for the development of bioartificial liver support systems. In this study the traditional centrifuge method was compared with the use of a Baylor Rapid Autologous Transfusion (BRAT) machine for isolating large quantities of porcine hepatocytes. After isolating hepatocytes, the methods were evaluated in terms of cell viability and yield per liver, proliferation over 7 days, and the effects on the cell cycle using the trypan blue exclusion test, conventional phase-contrast light microscopy, the lactate to pyruvate ratio, the leakage of lactate dehydrogenase (LD) and aspartate aminotransferase (AST), lidocaine clearance, albumin production, and flow cytometry. With the centrifuge method the mean cell viability was 92.5%, while with the BRAT method the viability was 95.9%. The minimal cell yields with the BRAT procedure were 7.3 x 10(9) for 250-ml centrifuge bowls and 2.8 x 10(9) for 165-ml bowls, which compares well with that found by other authors. Because the same initial procedures were employed in both methods the total hepatocyte yield per liver was comparable. Flow cytometry confirmed that the proliferation of hepatocytes was facilitated by oxygenation during the isolation procedure. The recovery of hepatocytes in culture following isolation was similar after either method. Daily microscopic investigation indicated that cytoplasmic vacuolization and granularities were present after either procedure and these disappeared following 3-4 days of culturing. Flow cytometry indicated that the hepatocyte cell cycle was similar after either method; at 7 days the profile indicated that the cells were still proliferating. Trends in the lactate to pyruvate ratio and the leakage of LD and AST indicated that the functional polarity of hepatocytes was regained after approximately 3 days. Lidocaine clearance at 4 days indicated that the cytochrome P450 system was active, while significant albumin production was apparent at day 5. The benefit of using BRAT technology in hepatocyte isolation lies in guaranteed sterility, convenience, speed, and the ability to oxygenate media and cell suspensions during the procedure.

Animals↗