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PubMed1963

EFFECT OF ETHAMBUTOL ON CYTOLOGY OF MYCOBACTERIUM SMEGMATIS.

Gale, Glen R. (Veterans Administration Hospital, Durham, N.C.) and Helen H. McLain. Effect of ethambutol on cytology of Mycobacterium smegmatis. J. Bacteriol. 86:749-756. 1963.-Electron microscopy showed the effects of the antimycobacterial drug, ethambutol [d-2,2'-(ethylenediimino)-di-1-butanol], on the cytology of Mycobacterium smegmatis. After 10 hr of exposure to the drug, cells no longer contained material which, in control cells, was closely associated with cellular division, possibly genetic substance. No marked changes were observed in other cellular structures. It was concluded that the drug may block one or more steps in the synthesis of pentose nucleic acid or deoxypentose nucleic acid, ultimately causing cessation of cellular division and loss of viability. These cytological findings were compatible with results of other investigators on the mode of action of this drug.

Antitubercular Agents↗
PubMed2004

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome.

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.

Cells, Cultured↗
PubMed2006

Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes.

Disaccharides such as sucrose and trehalose play an important role in stabilizing cellular structures during dehydration. In fact, most organisms that are able to survive desiccation accumulate high concentrations of sugars in their cells. The mechanisms involved in the stabilization of cellular membranes in the dry state have been investigated using model membranes, such as phosphatidylcholine liposomes. It has been proposed that the lyoprotection of liposomes depends on the depression of the gel to liquid-crystalline phase transition temperature (T(m)) of the dry membranes below ambient and on the prevention of membrane fusion by sugar glass formation, because both lead to leakage of soluble content from the liposomes. Since fusion is prevented at lower sugar/lipid mass ratios than leakage, it has been assumed that more sugar is needed to depress T(m) than to prevent fusion. Here, we show that this is not the case. In air-dried egg phosphatidylcholine liposomes, T(m) is depressed by >60 degrees C at sucrose/lipid mass ratios 10-fold lower than those needed to depress fusion to below 20%. In fact, T(m) is significantly reduced at mass ratios where no bulk sugar glass phase is detectable by Fourier transform infrared spectroscopy or differential scanning calorimetry. A detailed analysis of the interactions of sucrose with the P=O, C=O, and choline groups of the lipid and a comparison to published data on water binding to phospholipids suggests that T(m) is reduced by sucrose through a "water replacement" mechanism. However, the sucrose/lipid mass ratios necessary to prevent leakage exceed those necessary to prevent both phase transitions and membrane fusion. We hypothesize that kinetic phenomena during dehydration and rehydration may be responsible for this discrepancy.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

[Effect of water stress on the photosynthetic apparatus of plants and the protective role of cytokinins: a review].

Characteristics of photosynthetic apparatus (the pool of pigments and proteins; the activity of photosystems; the intensities of in vivo photoassimilation of carbon dioxide and in vitro activity of enzymes of carbon metabolism; leaf structure; chloroplast structure), undergoing changes under the conditions of water deficiency, have been reviewed. The protective role of cytokinins is due to their regulatory effects on the renewal of disrupted cellular structures, the condition of the stomata, and de novo synthesis and activation of proteins that are required for increasing plant resistance to water stress.

Cytokinins↗
PubMed1984

[Demonstration of the effects on the corneal epithelium of a mucomimetic collyrium. Study using scanning microscopy].

Ocular surface drying was studied in rabbits using an experimental model of blinking interruption. This model produces a discontinuity in the pre-corneal tear film after a few minutes of exposure to air. Changes in superficial epithelial cell structure were studied using a scan microscope. A decrease of micro-folds and an increase in epithelial cell desquamation were observed. Prolonged exposure to air produced similar cellular lesions in deeper layers. The instillation of a single mucomimetic eye-drop containing chondroitin sulphate prevented total interruption of epithelial humidification, kept the epithelial surface intact and preserved normal cellular structure.

Animals↗
PubMed2004

The novel lectin KM+ detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum.

KM+ is a D(+)mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+)mannose, but not D(+)galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-alpha1,3-[man-alpha1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosyl-glycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates.

Animals↗
PubMed2004

Diffusion and reaction-diffusion in fast cellular flows.

Cellular structures, as the rolls generated by Rayleigh-Bénard instability, have always been an important topic in nonlinear science. The diffusion of a passive scalar in a given steady cellular flow becomes an interesting question in the limit of a large Péclet number, often realistic. The main result there is that the effective diffusion is somewhere in between the molecular diffusion and the "turbulent" diffusion. A new added twist to this is the reaction-diffusion case, where the front speed is the laminar propagation velocity (without flow) times the Péclet number to the power 1/4. I refine this last result and give the behavior of the prefactor in the Zel'dovich limit of a narrow reaction zone.

Journal Article↗
PubMed1983

Selective aberration and pigment loss in melanosomes of malignant melanoma cells in vitro by glycosylation inhibitors: premelanosomes as glycoprotein.

We have found that glucosamine (1 mg/ml) or tunicamycin (0.2-0.4 micrograms/ml), specific inhibitors of lipid carrier-dependent glycosylation of protein, when added to cultured B-16 melanoma cells produce a marked loss of pigmentation, accompanied by distinctive biochemical as well as ultrastructural aberrations in their melanogenic compartments. Electron microscopic analysis shows that these newly induced unpigmented cells form uniquely altered melanosomes containing little or no melanin, although their population is not substantially reduced. Within the melanogenic compartments, selective aberration of melanosomes is seen, that is, deformity, bulging, and segregation of their interior membrane, as well as the intramelanosomal formation of irregularly concentric lamellar structure. No apparent structural deformity of Golgi apparatus, Golgi-associated endoplasmic reticulum of lysosome (GERL), and coated vesicles has been observed. Further, no substantial alteration is seen in mitochondria or other cellular structures. Quantitative analysis of altered and nonaltered melanosomes has revealed that the ratio of altered premelanosomes to the total number increases to 44% in glucosamine-treated cells and to 99.5% in tunicamycin-treated cels. Compared to 13% in the control. Electron microscopic dopa reaction has also revealed that these altered melanosomes seem to exhibit a weakly positive or a negative dopa reaction in both glucosamine- and tunicamycin-treated melanoma cells although a number of dopa-positive altered melanosomes are still seen. However, GERL and coated vesicles show no apparent decrease in dopa reaction. It may be concluded that glycoprotein synthesis is integral to the formation of normal melanosomes and to their specific melanizing function, which could be impaired by inhibition of the synthesis of asparagin-linked mannose-containing sugar chains. This results in retrogressive changes in the premelanosomal tyrosinase during its maturation process.

Animals↗
PubMed1999

New experimental model to study the bone interface of endosseous implants: an in vitro three-dimensional model of cell culture.

To set up a three-dimensional culture model of endosseous implant material-osteoblast, titanium (Ti) discs were placed onto confluent cellular layers of human fetal osteoblasts that were cultured in calcifying conditions. Follow-up observations of living cells with phase-contrast microscopy and histological observations of the cell-Ti disc interface with ordinary microscopy and transmission electron microscopy were used to monitor and verify the formation of three-dimensional cellular structures at the osteoblast-Ti disc interface. These methods were further used to study osteoblast cell behavior during the early stage of bone healing in this three-dimensional cellular model. The follow-up observation showed that, upon the placement of the Ti disc, cells migrated from the cellular layer and attached to the rim of the disc, forming cell bridges. Cells continued to attach and orient throughout the culture time. A cellular multilayer at the osteoblast-Ti interface was formed, which resulted in the three-dimensional model. Furthermore, transmission electron microscopy observation showed that there was a mineralizing process at the osteoblast-Ti disc interface just like bone, which confirms the reality of this model. Histological observations with conventional microscopy demonstrated the interfacial relation between biomaterial and cells. If osteoblasts were replaced by other types of cells, this model could be used to study the other kinds of interfaces.

Cell Adhesion↗
PubMed1987

Alpha-actinin-containing aggregates in transformed cells are highly dynamic structures.

Normal rat kidney cells infected with a Rous sarcoma virus (strain LA23) were used to study the dynamics of alpha-actinin-containing aggregates in transformed cells. Experiments were performed by microinjecting living cells with iodoacetamidotetramethylrhodamine alpha-actinin and allowing the fluorescent analogue to incorporate into cellular structures. Subsequent time-lapse recording indicated that the alpha-actinin-containing aggregates can undergo rapid formation, movement, and breakdown. In addition, experiments using the photobleaching recovery technique indicated that alpha-actinin molecules associated with the aggregates have a very high rate of exchange, whereas those associated with adhesion plaques in normal cells exchange much more slowly. The dynamic properties of alpha-actinin-containing aggregates may be closely related to the changes in cellular behavior upon oncogenic transformation.

Actinin↗
PubMed1989

Freeze-fracture cytochemistry: review of methods.

"Freeze-fracture cytochemistry" encompasses a diversity of recently developed techniques in which freeze-fracture and cytochemistry are combined. Cytochemical labeling may, in principle, be integrated into one of three basic points in the standard freeze-fracture procedure; 1) before the specimen is frozen, 2) after it has been fractured, or 3) after it has been platinum shadowed and/or carbon coated. Visualization of the labeled cellular structures can be achieved by a variety of different methodologies. For example, the markers (usually colloidal gold particles) may be viewed embedded within a replica, or attached to it via fragments of membrane (or other cellular components). Sectioning is a central strategy in a number of techniques, either in combination with or in place of replication. The different combinations of methods that have been devised are not, for the most part, alternative ways of arriving at the same result; each provides quite distinct information about specific classes of membrane component or other structure in the cell. The purpose of this review is to present, within a single article, a systematic survey of the full range of techniques currently available in freeze-fracture cytochemistry. Emphasis is placed on explaining the principles underlying the methods and on illustrating their applications. With the success recently achieved, freeze-fracture cytochemistry has moved from the phase of experimental development to a position in which it may be expected increasingly to make significant contributions across a wide spectrum of problems in cell and membrane biology.

Animals↗
PubMed1999

Targeting of cytoskeletal linker proteins to focal adhesion complexes is reduced in fibroblasts adhering to laminin-1 when compared to fibronectin.

Cellular interactions with the extracellular matrix determine to a large extent cell behavior, including cell migration. These interactions take place at specialized cellular structures, the focal adhesions, which have a substrate-specific morphology. To determine the molecular and functional relevance of this observation, the composition of isolated focal adhesions developed by fibroblasts adhering to fibronectin or laminin-1 was analyzed by indirect immunofluorescence and immunoblotting with or without stabilization of the structures by cross-linking. In the absence of cross-linking, integrins, talin, vinculin and, to a lower extent, paxillin remained associated with the focal adhesions formed on both substrates, indicating a tight association of these proteins with the extracellular matrix support. By contrast, alpha-actinin, FAK, and actin were apparently loosely maintained within focal adhesions and were found associated to these structures only after stabilization by cross-linking. Interestingly, although both substrates induced clustering and aggregation of all these proteins, their relative concentration, with the exception of alpha-actinin, was lower within the focal adhesions formed on laminin-1 than in those formed on fibronectin. Moreover, as assessed in migration assays, the locomotory speed of fibroblasts was higher on laminin-1 than on fibronectin. Altogether these results indicate that integrins involved in cellular interactions with fibronectin or laminin-1 trigger the formation of focal adhesion structures which differ by molecular organization, concentration in several adhesion plaque components, and function.

Actinin↗
PubMed2005

Electron microscopy of cultured angiogenic endothelial cells.

Angiogenesis is a multi-step process involving migration, proliferation, and a specific spatial arrangement of endothelial cells. On the basis of a model of cultured microvascular endothelial cells derived from the bovine corpus luteum, all stages of in vitro angiogenesis as well as intussusceptive remodeling were characterized by scanning and transmission electron microscopy. To preserve the delicate three-dimensional cellular structures for electron microscopy, modified processing techniques for both transmission and scanning electron microscopy including micro-corrosion casting of cultured cells were established. The detailed results on morphological alterations and cellular interactions confirmed and complemented earlier studies of in vitro angiogenesis. Electron microscopy proved to be an efficient tool for detection and supervision of all major endothelial differentiation processes resembling in vivo conditions that are generally considered important in a realistic in vitro model of angiogenesis: occurrence of function-related cellular junctions; development of specific surface features indicating cellular polarity; production of extracellular matrix material; mechanisms leading to the formation of an internal lumen; specific spatial arrangement of endothelial cells within capillary-like networks; detachment of apoptotic cells as well as intussusception of specific cells within the course of vascular remodeling. The abundance of quickly available information provided by electron microscopic approaches may be useful for subsequent, e.g., biochemical or molecular, studies and thus delivers important controls for further experimental designs.

Animals↗
PubMed1998

[Juvenile pleomorphic parotid adenoma of embryonal structure].

Juvenile pleomorphic adenoma of the parotid gland represents an extremely rare tumour entity and is comparable to congenital tumours of the salivary glands concerning its embryonal structure. The clinical detection of the tumour in a 7-year-old girl does not exclude that the tumour had developed either earlier or immediately after the birth. The high cellularity and the evidence of primitive epithelial and myoepithelial cellular structures do not justify its classification as a malignant tumour. However the presence of embryonal tissue structures associated with the end of the third month of embryogenesis is characterized by more solid cell formations in partly verticulate arrangement. The absence of further differentiation into lobular structures and differentiated duct or acinic cell formations may be due to cell arrest. Differential diagnosis of juvenile pleomorphic adenoma must distinguished it from other congenital salivary gland tumours (e.g. congenital basal cell adenoma, hybrid basal cell adenoma-adenoid cystic carcinoma, sialoblastoma, salivary gland anlage tumour.

Adenoma, Pleomorphic↗
PubMed2002

125I-labeled galanin binding sites in congenital innervation defects of the distal colon.

Neuropeptides have turned out to be promising new parameters, in addition to the routinely performed histochemical diagnosis, of Hirschsprung's disease (HD). Studies of the peptidergic innervation of the affected intestinal segment of patients with HD have demonstrated a marked reduction in the density of several neuropeptide-containing nerve fibers. The frequency of nerve fibers storing the neuropeptide galanin (GAL) was found to be unchanged or slightly reduced in HD, but nothing is known about the occurrence of GAL receptors. In this study, in vitro receptor autoradiography using (125)I-labeled GAL and GAL immunofluorescence have been performed on frozen tissue sections from colon biopsies of 10 patients diagnosed with HD, 8 patients with intestinal neuronal dysplasia (IND B) and 20 patients with chronic obstruction but normal innervation. Binding sites were mainly detected in the mucosal and muscular layer, in acetylcholinesterase-positive nerve fiber bundles and ganglia within the submucosal layer and in close association to blood vessels. An increased population of GAL receptor positive, parasympathetic nerve fibers was seen in the aganglionic segment of HD as compared to controls and IND B. In contrast, GAL immunostaining which was unchanged in HD revealed a significant lack of GAL-positive structures in IND B colon biopsies. Colocalization of GAL and GAL binding sites was only observed in thick nerve fibers in the submucosa. The presence of GAL binding sites in different cellular structures suggests an involvement of GAL in various physiological functions of the gastrointestinal tract.

Adolescent↗
PubMed2006

Behavior of Trichoderma reesei hydrophobins in solution: interactions, dynamics, and multimer formation.

Filamentous fungi utilize small amphiphilic proteins called hydrophobins in their adaptation to the environment. The hydrophobins are used to form coatings on various fungal structures, lower the surface tension of water, and to mediate surface attachment. Hydrophobins function through self-assembly at interfaces, for example, at the air-water interface, and at fungal cellular structures. Despite their high tendency to self assemble at interfaces, hydrophobins can be very soluble in water. To understand the mechanism of hydrophobin self-assembly, in this work, we have studied the behavior of two Trichoderma reesei hydrophobins, HFBI and HFBII in aqueous solution. The main methods used were Förster resonance energy transfer (FRET) and size exclusion chromatography. A genetically engineered HFBI variant, NCys-HFBI, was utilized for the site-specific labeling of dyes for the FRET experiments. We observed the multimerization of HFBI in a concentration-dependent manner. A change from monomers to tetramers was seen when the hydrophobin concentration was increased. Interaction studies between HFBI and HFBII suggested that at low concentrations homodimers are preferred, and at higher concentrations, the heterotetramers of HFBI and HFBII are formed. In conclusion, the results support the model where hydrophobins in aqueous solutions form multimers by hydrophobic interactions. In contrast to micelles formed by detergents, the hydrophobin multimers are defined in size and involve specific protein-protein interactions.

Crystallography, X-Ray↗
PubMed2004

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy.

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.

Animals↗
PubMed1988

Expression of a high molecular weight mucin-type glycoprotein in human colon cancer as defined by monoclonal antibody 1D3.

Murine monoclonal antibody 1D3 recognizes a high molecular weight acidic mucin restricted to the epithelium of normal colonic mucosa and goblet cells. Of 72 colonic carcinoma specimens examined, 29 were found to have detectable level of 1D3 antigen by an indirect immunoperoxidase staining assay on fixed, paraffin-embedded tissue sections. In some specimens a focal staining pattern was observed, while in others 50-90% of tumor cells were stained. Of 28 cases having adjacent normal mucosa, all 28 showed intense staining reaction in the normal mucosa and goblet cells despite the fact that 18 of the tumors were unstained. One of 1 colonic diverticulosis, 2 of 2 ulcerative colitis, 3 of 3 villo-glandular polyps, 19 of 20 adenomatous polyps and 17 of 19 hyperplastic polyps were also stained heavily for the 1D3 antigen. Colonic carcinomas displayed a range of staining patterns and a great degree of antigenic heterogeneity. Well-differentiated tumors characterized by typical goblet cells were almost always positive (10 of 12). As cellular structure became disorganized, as in moderately-differentiated tumors, about 33% of the tumors (17 of 51) stained for 1D3 antigen. As the tumor became more invasive with further disorientation of cellular features, as in poorly differentiated tumors, very few specimens (2 of 9) were positive. It was apparent that with the progression of de-differentiation there was a gradual loss of 1D3 antigen in human colonic tumors.

Antibodies, Monoclonal↗