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The hepatic lesion in protoporphyria (PP): preliminary studies of haem metabolism, liver structure and ultrastructure.

Five unrelated patients with protoporphyria (PP) had diagnostic liver biopsies performed to assess the degree of liver damage. The porphyrin content of the liver was quantitated and characterized and liver damage was assessed. Ultraviolet (UV) microscopy was performed in each case. Liver structure was assessed by light, polarization and electron microscopy. In 3 patients the liver was visualized directly before biopsy through a peritoneoscope. Liver damage ranged from minimal cell necrosis to portal fibrosis; the latter was observed in a 27-year-old sib of a patient (M.I.) who had died, aged 29, 3 years previously in liver failure from PP-related cirrhosis. Liver tissue from the latter patient which was obtained at the time of autopsy was re-examined by light and polarization microscopy. Hepatic pigment deposits, thought to be lipofuscin, showed birefringence on polarization microscopy in two cases, one of them being patient M.I. with PP-cirrhosis. Liver fluorescence on UV microscopy was centrizonal, punctate, faded rapidly and was easily distinguishable from that seen in porphyria cutanea tarda (PCT). The porphyrin content of the liver tissue in biopsied patients was between 5 mug and 80 mug, and in the autopsy case 1600 mug protoporphyrin/g wet weight liver, and on thin layer chromatography only dicarboxylic porphyrins were demonstrable. Hepatic cytochrome P-450 levels in protoporphyria were within normal range. Vmax and Km for aminopyrine-N-demethylation and benzpyrene hydroxylation did not differ significantly from our findings in PCT, variegate porphyria in remission and in non-porphyric controls. However, the activity of hepatic delta-aminolaevulinic acid (ALA) synthetase was significantly enhanced in 2 of the 3 patients in whom this measurement was performed.

Adult↗

[Study of human chromosomes using fluorescence technics].

Fluorescent techniques permitted us to draw up a system of banding of human chromosomes, using quinacrine derivatives. This procedure may be used to measure the density of the chromosomes. Recently, other techniques which use various fluorochromes after denaturation, have been developed. The latter, which use acridine orange, give remarkably reliable and reproducible results. These techniques are particularly useful for the analysis of structural changes in the chromosomes. Their use is illustrated by a certain number of observations where they permitted the authors to determine the type of structural change and identify the chromosomes involved. The method of constitution and significance of the fluorescent bands is very controversial. It is not clearly known whether the fluorochromes combine with DNA or with protein bound to the latter. In spite of this uncertainty, the existence of heterogeneity in the structure of the chromatids at the metaphase provides new information for the understanding of chromosome organisation.

Acridines↗

[Oncocytes--a system of serotoninocytes].

Oncocytes, irrespectes, irrespective of their localization in this or that organ, possess only one active function inherent in all cells of the given type; this function depends on serotonin cantained therein. Consequently, oncocytes are not afunctional perishing cells, as was thought up to this time, but represent a group of serotoninocytes appearing in the form of a widely spread in the organism of the diffuse peripheral neuro-endocrine system, the activity of which is associated with the function of the givern biogenic monoamine.

Esophagus↗

Localization of polycyclic hydrocarbon carcinogens in the lung following intratracheal instillation in gelatin solution.

The deposition and localization of benzo(a)pyrene (BP) suspended in a gelatin-0.9 percent NaCl solution was studied in hamster lungs by ultraviolet fluorescence microscopy. BP was deposited primarily in the alveolar region of the lung. Although large numbers of BP-filled macrophages were seen in the upper airways by 24 hr after an instillation, little BP appeared to penetrate into the bronchial epithelium. The intratracheal instillation of polycyclic hydrocarbons in a gelatin-0.9 percent NaCl solution appears to be a useful model when it is desired to deliver the carcinogen dose to the peripheral lung.

Animals↗

Efficiency of lipofection of adherent cells is limited by apoptosis.

Stability of gene expression and transfection efficiency plays the main role in the application of gene transfer method. In somatic cell gene delivery, expression of the gene product is limited by the function of the cell to which it is delivered. In the present study analyzing the lipofected adherent cells, we have shown that lower level of transgene: beta-galactosidase activity at later time period correlated with decrease in cell viability, which was shown to be due to apoptosis. Apoptosis following DNA uptake occurred only when DNA was present during lipofection.

Apoptosis↗

[Study of the structure of colonies of active and inactive Actinomyces parvullus variants using luminescence and scanning microscopy].

The colony structure of the active and inactive proactinomycete-like variants of Actinomyces parvullus producing actinomycin D was studied with luminescent and scanning microscopy. Clear differentiation of the colony profile was shown by the structure and functions of the mycelium layers. A zone of active synthesis and accumulation of the antibiotic was observed in the colonies of the active variant in the upper part of the substrate mycelium with reddish-yellow self luminescence in UV light and characteristic close hyphae "cemented" by the intracellular substance. Formations of the granule type were often noted on the hyphae of that layer. The layer of the aerial mycelium was loosely connected with the substrate mycelium and consisted of sporophores and spore chains partially broken into single spores. The colonies of the inactive proactinomycete-like variant had a slightly differentiated profile with a sponge-like structure, no zones of the antibiotic synthesis being found. The presence of the intracellular substance was observed in the upper part of the colony supersubstrate mycelium.

Dactinomycin↗

Effects of FK506 and cyclosporin A on cytokine production studied in vitro at a single-cell level.

Mononuclear cells obtained from human blood were mitogen or antigen activated in vitro in the presence or absence of FK506 or cyclosporin A (CsA). Cytokine production was studied at a single-cell level by ultraviolet (UV) microscopy of fixed permeabilized cells using cytokine-specific monoclonal antibodies (mAb). Phenotypic characterization of the monokine-producing cells was achieved by two-colour immunofluorescent staining. Cytokine production after antigen activation with Staphylococcus aureus enterotoxin A (SEA) was significantly reduced. FK506 or CsA inhibited SEA-induced tumour necrosis factor-alpha (TNF-alpha) production both in monocytes (P less than 0.01) and in lymphocytes (P less than 0.001), at a drug concentration of 1-25 ng/ml for FK506 and 100-500 ng/ml for CsA. Lymphocyte synthesis of interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and TNF-beta after SEA activation was also significantly reduced by either of the drugs. In contrast, endotoxin-induced monokine production (TNF-alpha and IL-6) after lipopolysaccharide (LPS) stimulation was unaffected by FK506 or CsA even when added in concentrations as high as 1000 ng/ml. When the cells were stimulated by phorbol ester (phorbol 12-myristate 13-acetate, PMA) plus calcium ionophore (ionomycin), FK506 and CsA inhibited, in a dose-dependent manner, the production of IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha. The 50% inhibitory concentration (IC50) for FK506 or CsA on the cellular synthesis of the various cytokines varied between 0.6 and 1.0 ng/ml and 20 and 60 ng/ml, respectively. Further stimulation by addition of anti-CD28 mAb to the cultures resulted in an augmented IL-2 and IFN-gamma production which was resistant to both FK506 and CsA. This report delineates extensive similarities between the two drugs in mechanisms of immunosuppression by blockade of identical interleukin production. Depending on the mode of cell activation the two drugs inhibited not only cytokine production in lymphocytes but also antigen-induced monokine (TNF-alpha) production in macrophages, although the optimal immunomodulatory effect of FK506 was achieved at a concentration approximately 50-fold lower than that of CsA.

Antigens, CD↗

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole.

The susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole (TZ) was examined. The minimal bactericidal concentration (MBC) of TZ against the mobile spirochetes was >128 microg/ml at 37 degrees C in micro-oxic atmosphere when incubated for 14 days. TZ significantly reduced the conversion of mobile spirochetes to cystic forms during incubation. The MBC for older (10-months-old) cysts at 37 degrees C in a micro-oxic atmosphere was >0.5 microg/ml, but >0.125 microg/ml for young (1-day-old) cysts. Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that, when the concentration of TZ was > or = MBC, the contents of the cysts were partly degraded, core structures did not develop inside the young cysts, and the amount of RNA in these cysts decreased significantly. When cysts were exposed to TZ, both the spirochetal structures and core structures inside the cysts dissolved, and the production of blebs was significantly reduced. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of TZ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.

Anti-Bacterial Agents↗

Effect of beta-mercaptoethanol or epidermal growth factor supplementation on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle.

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.

Animals↗

[The traces of smokeless gunpowder].

The experimental investigation resulted in detection of new specific and characteristic signs of smokeless powder. Additional tests for determination and differential diagnosis of smokeless powder variants were suggested.

Diagnosis, Differential↗

[An analysis of the mechanisms of interpopulation interaction of Yersinia with Tetrahymena pyriformis infusorians at the cellular and subcellular levels].

The mechanisms of interaction between the populations of Yersinia and T. pyriformis have been analyzed on the cellular and subcellular levels. As shown in this investigation, Yersinia, when phagocytized by T. pyriformis, may undergo morphological changes, remain unchanged and also multiply, destroying the host cell in the process.

Animals↗

Identification and purification of NK cells with lysosomotropic vital stains: correlation of lysosome content with NK activity.

The lysosome content of lymphocytes has been analyzed with lysosomotropic vital stains and the fluorescence-activated cell sorter (FACS). Large granular lymphocytes (LGL), which account for virtually all natural killing activity in peripheral blood, are quantitatively different from small lymphocytes (SL) in this respect. LGL obtained by Percoll gradient density centrifugation accumulate more of the lysosomotropic vital dyes than SL do, staining with either neutral red or mepacrine (quinacrine). Furthermore among the LGL-rich, low density lymphocyte population highly, granulated cells can be separated from less granulated ones by mepacrine staining and FACS. Thus, separated highly granulated LGL express very high natural killing, whereas the less granulated low density large lymphocytes do not kill.

Cell Separation↗

[The intraoperative diagnosis of cardiomyocyte damage].

The morphology of myocardial biopsies taken in the course of the operative valve prosthesis is studied light and electron microscopically with the use of the fuchsinorrhagic index and tracer test. The dynamics and morphogenesis of cardiomyocyte injury depend upon their initial state. The myocardial changes characteristic of mitral valvular disease progress during the cardiac arrest. Besides that, observed the destabilization of membrane systems, tracer penetration of the injured cells and inner organelles, the fall of redox potential of mitochondria, the disturbance of the acidic hydrolases compartmentalization, the accumulation of multilamellar bodies indicating the membrane alteration. The combination of these changes determines the myocardial contractility in the period of rehabilitation and raises the risk during the prolonged operations. The suggested tests simultaneously give structural and metabolic estimation of the cardiomyocyte organelles in respect to the prognosis of irreversible injuries.

Biopsy↗

Light and electron microscopic changes in sural nerves in Ethiopian diabetics.

The light and electron microscopic morphological changes in sural nerve biopsies from 32 Ethiopian diabetics and 4 malnourished non-diabetic subjects were studied in order to determine the role of malnutrition in the development of diabetic neuropathy. The most prominent finding in diabetics with clinical neuropathy was reduction in the density of myelinated axons of all diameters especially the large ones. Affected nerves showed abundant intraneural collagen, and evidence of nerve regeneration in some cases. Demyelination, though present, was less marked. Changes in the vasa nervorum were seen in only one case. The morphological changes correlated with the duration and severity of the diabetes and with nerve conduction velocity. No changes were seen in malnourished non-diabetics and diabetics without clinical neuropathy, thus suggesting that malnutrition by itself does not contribute significantly to the development of diabetic neuropathy. Axonal degeneration appears to be the predominant feature in diabetic neuropathy.

Adolescent↗