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PubMed1987

Cytoarchitecture and the patterning of fushi tarazu expression in the Drosophila blastoderm.

In the Drosophila embryo at the blastoderm stage, the segmentation gene fushi tarazu (ftz) is expressed in a seven-banded pattern. The generation of this pattern, like many other segmentation gene expression patterns, coincides with the formation of cell membranes around the blastoderm nuclei. To test the role of cellularization in resolving the banded ftz pattern, we used cytoskeletal inhibitors (colcemid and cytochalasin B) to block cellularization. We found that banded ftz RNA and protein patterns can form without cellular structure. We also tested the importance of rapid degradation of the ftz RNA, using cycloheximide to block degradation. RNA degradation is essential to maintain the banded ftz pattern in a syncytium, but is not required to maintain the pattern in a cellularized embryo. A latticework of cytoskeletal microtubules that forms during cellularization appears to be a key component in localizing the ftz mRNA. We conclude that RNA degradation and cellular structure normally work together to localize ftz RNA to its sites of synthesis.

Animals↗
PubMed1982

[Role of membrane colchicine binding proteins in the transmission of prolactin message to casein genes in the rabbit mammary gland].

Previous work demonstrated that tubulin binding drugs specifically inhibit the capacity of prolactin to initiate casein and DNA synthesis in the mammary cell. It was concluded that microtubules or other tubulin containing cellular structures were involved in the transmission of the prolactin message to genes. In the present work, it is shown that griseofulvin, an antimitotic drug which alters microtubule structure and function, does not prevent prolactin actions. Autoradiographic studies showed that [3H]colchicine binds preferentially to plasma and Golgi membranes in the mammary cell. Short term cultures of mammary explants with [3H]colchicine demonstrated that the labelled drug binds to membranous cellular structures which were isolated from explants at the end of the culture. Fractions containing plasma and Golgi membranes contained the highest amount of radioactivity. Solubilisation of the membranes by Triton X-100 dissociated the [3H]colchicine from the prolactin receptors as judged by a chromatography of the soluble fraction on a Sepharose 6 B column. On the column, the labelled colchicine remains associated with a molecular entity which may be free tubulin. In all cases, the binding of [3H]colchicine was greatly attenuated by an excess of unlabelled colchicine but was only slightly affected by the competition with lumicolchicine. These results suggest that mammary membranes contain tubulin and that binding of drugs to this molecule inhibits the generation of the prolactin second messengers eliciting the hormonal actions in the mammary cell. This also suggests that microtubules are probably not involved in the mechanism of prolactin action.

Animals↗
PubMedDate not supplied

Quantitation of GFP-fusion proteins in single living cells.

The green fluorescent protein (GFP) has revolutionized cell biology. The ability to observe genetically encoded fluorescently tagged fusion proteins in intact cells has made virtually any biological process amenable to investigation in living cells. However, most in vivo imaging studies are qualitative and little information about the number of fluorescently labeled molecules observed in a cell or a cellular structure is available. This deficiency severely limits the interpretation of imaging experiments and it impedes the application of in vivo imaging methods for biophysical purposes. Here we describe a simple method for the quantitative determination of the number of GFP-tagged molecules in cellular structures in single living cells. The method is based on the use of rotavirus-like particles containing a known number of GFP molecules as an internal calibration standard during in vivo imaging. We have applied this method to estimate in single living cells the number of fluorescent transcription factor molecules on RNA polymerase I and polymerase II genes. In addition, we have estimated the number of molecules for several proteins in subnuclear compartments and in exocytic vesicles. VLP-GFP calibration is a simple, convenient, rapid, and noninvasive method for routine quantification of GFP-labeled molecules in single, living cells.

Animals↗
PubMed1990

Structural and cellular characterization of solvent-casted polyurethane membranes.

Fibroblastic cell attachment and growth characteristics of different polyurethane (PU) films were tested. These films were prepared by a classical solvent-casting procedure. By changing the composition and the type of casting solution (i.e. tetrahydrofurane, dioxane, dimethyl formamide-tetrahydrofurane, tetrahydrofurane-dioxane, etc.) PU films with different physical and chemical bulk and surface structures were obtained. Structural properties of these films were investigated by scanning electron microscopy, equilibrium swelling experiments and contact-angle studies. In stationary cell culture tests, a model cell-line, i.e. baby hamster kidney (BHK) were used. Thus the effects of structural properties on the cell behaviour were investigated. The results demonstrate that it is possible to achieve different cell responses by changing the preparation conditions of the films. While the cell attachment is excellent on porous PU surfaces, the others showed similar adhesion. Better proliferation of BHK cells was obtained with PU films prepared from dioxane solution.

Animals↗
PubMed1994

Rearrangement of mRNAs for costamere proteins during costamere development in cultured skeletal muscle from chicken.

Mature skeletal myofibrils are surrounded by costameres, ribs of metavinculin, vinculin, intermediate filaments, and other proteins that connect the myofibril to the extracellular matrix. Costameres have recently been shown to be the sites at which the forces generated by the myofibril are transduced laterally into the extracellular matrix. We observed costameres developing in cultured skeletal muscles, grown in micromass culture from cells taken from embryonic chicken leg. We detected proteins by immunofluorescence and mRNA by in situ hybridization. Antibody and probe signals were imaged by laser scanning confocal microscopy. Antibody to vimentin protein is first detected in stripes in register with the Z line of the myofibril, at approximately day 12 after fusion; soon thereafter probe to vimentin mRNA is also detected in the same stripes. Optical sections indicate that vimentin mRNA and protein are very close, no more than 0.1 mm apart and possibly in immediate contact. Antibody to vimentin is detected in stripes only in cells that twitch spontaneously. Antibodies and probes to desmin and vinculin protein and mRNA are next detected in stripes of the same periodicity, at approximately day 17 after fusion. Vinculin protein (but not mRNA) is detected at focal contacts much earlier in development. Controls for bleed through of fluorescence, RNase H sensitivity, hybridization without probe, and binding to the myofibril all gave appropriate results. Probes to glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme, stained diffusely and did not associate with the myofibril. These results show that components of the costamere arrive at the structure in a defined sequence, and that mRNA organization is a conspicuous, precise and temporally controlled aspect of costamere development. These results may have wider implications. In these cells, some mRNAs are positioned with submicrometer precision in space and differentially over time. Particular mRNAs differ in the time and place of such positioning. This implies both that cellular structures provide physical cues for such positioning and that mRNA contains information that interacts with such cues in a message-specific manner. If such precision in mRNA location is found in other somatic cells, it could have significant implications for the ways in which cells generate and maintain cellular structures.

Animals↗
PubMed2003

Structural and cellular architecture of conjunctival lymphoid follicles in the baboon (Papio anubis).

Conjunctival lymphoid follicles (CLFs), present in normal individuals, undergo hyperplasia upon conjunctival infection by a specific array of pathogens; infection-associated enlargement of draining preauricular lymph nodes suggests that conjunctival follicles participate in the afferent limb of acquired immune responses for the ocular surface. The present study was performed to delineate the structural and lymphoid anatomy of CLFs in the baboon (Papio anubis), a non-human primate conjunctival model with close similarity to the human. Conjunctiva from both eyes, along with mesenteric lymph node, spleen, tonsil, and ileum controls were harvested from ten baboons at necropsy, and studied by histochemical and immunohistochemical methods. Baboon conjunctival follicles were identified as dense oval collections of leukocytes in the substantia propria with infiltration into a thinned overlying conjunctival epithelium. Goblet cells were universally absent, the overlying mucin layer was attenuated, and the follicle-associated epithelium (FAE) demonstrated comparatively diminished alkaline phosphatase expression. The basement membrane overlying each follicle appeared discontinuous. CD4-positive T lymphocytes were distributed in parafollicular areas and to a lesser degree in follicle germinal centers. B lymphocytes formed the predominant cell in follicles, and also heavily infiltrated the FAE. B cell IgM expression was prominent in germinal centers, while IgD staining occurred in a horseshoe-shaped distribution in the follicle mantle zone. Although B cell IgA expression was noted in the non-follicular conjunctiva, IgA expression was inconspicuous within conjunctival follicles. S-100- and CD1a-positive dendritic cells were found in FAE, while fascin-positive mature dendritic cells appeared in the deeper areas of each follicle. CD68-positive macrophages were dispersed throughout the follicles. CD35-positive follicular dendritic cells were observed only in germinal centers. CLFs appear highly organized consistent with a role in the adaptive immune response to conjunctival pathogens.

Animals↗
PubMed2005

Identification of novel clock-controlled genes by cDNA macroarray analysis in Chlamydomonas reinhardtii.

Circadian rhythms are self-sustaining oscillations whose period length under constant conditions is about 24 h. Circadian rhythms are widespread and involve functions as diverse as human sleep-wake cycles and cyanobacterial nitrogen fixation. In spite of a long research history, knowledge about clock-controlled genes is limited in Chlamydomonas reinhardtii. Using a cDNA macroarray containing 10 368 nuclear-encoded genes, we examined global circadian regulation of transcription in Chlamydomonas. We identified 269 candidates for circadianly expressed gene. Northern blot analysis confirmed reproducible and sustainable rhythmicity for 12 genes. Most genes exhibited peak expression at the transition point between day and night. One hundred and eighteen genes were assigned predicted annotations. The functions of the cycling genes were diverse and included photosynthesis, respiration, cellular structure, and various metabolic pathways. Surprisingly, 18 genes encoding chloroplast ribosomal proteins showed a coordinated circadian pattern of expression and peaked just at the beginning of subjective day. The co-regulation of genes bearing a similar function was also observed in genes involved in cellular structure. They peaked at the end of the subjective night, which is when the regeneration of cell walls and flagella in daughter cells occurs. Expression of the chlamyopsin gene, which encodes an opsin-type photoreceptor, also exhibited circadian rhythm.

Animals↗
PubMed2002

Evidence for consistent patterns between flavonoid structures and cellular activities.

A wide variety of plant-derived compounds, including the polyphenolic flavonoids, is present in the human diet or is consumed for medicinal reasons. Epidemiological and animal studies tend to suggest a protective effect of flavonoids against cardiovascular diseases and some types of cancer. Although flavonoids have been studied for about 50 years, the cellular mechanisms involved in their biological activity are still largely unknown. Antioxidant properties of the flavonoids have been postulated as a mechanism for putative protective effect against cardiovascular disease. Nevertheless, these properties alone are not sufficient to explain the anti-carcinogenic potential of these polyphenols. The mechanisms by which the molecules interact with cells or are absorbed by them are very important for determining the intracellular concentration and distribution of the metabolites to internal organs. With the exception of the cells lining the gastrointestinal tract, all other cells in the body are only exposed to flavonoid metabolites and degradation products. No previous studies have addressed this aspect of cellular exposure, except for some methylated metabolites. Within the last decade, reports on flavonoid activities have been largely associated with enzyme inhibition and anti-proliferative activity. From our recent work on the human colon cancer cell line HT29 and comparison with published studies, structure-function relationships demonstrate that antioxidant, enzyme inhibitor or anti-proliferative activities are dependent on particular structure motifs. The present review also presents a summary of mechanistic data on a few elected compounds.

Animals↗
PubMed2003

[Protecting effects of p44/42 MAPK signal transduction pathway on hepatocytes in ischemic preconditioning].

OBJECTIVE: To investigate the significance of PKC and p44/42 mitogen-activated protein kinase (MAPK) signal transduction in ischemic preconditioning (IP). METHODS: Through liver cell IP models, PKC inhibitor and MEK inhibitor were utilized to analyze the phosphorylation level of p44/42 MAPK and cell viability was also observed. Rat liver IP models were established which were treated with various drugs. Then the phosphorylation level of p44/42 MAPK in vivo and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) concentrations were detected. And cellular structures were observed under light microscopy. RESULTS: Similar results were obtained in vivo and in vitro IP models. Compared with the ischemia reperfusion (IR) group in vivo, the phosphorylation level of p44/42 MAPK was obviously increased in IP treated rats (q = 27.217, P < 0.01), and the cellular structure injured slightly. The concentrations of serum ALT and AST in IP group were significantly lower than those in IR group (281.0 U/L +/-35.6 U/L vs 762.8 U/L +/-130.5 U/L and 407.7 U/L +/-73.7 U/L vs 820.9 U/L +/-111.3 U/L, P < 0.01). However, opposite changes were found in PKC and MEK inhibited groups, when compared to IP group. The phosphorylation level of p44/42 MAPK was obviously decreased, the liver tissues injured evidently, and the concentrations of serum ALT and AST (645.61 U/L +/-90.4 U/L, 678.6 U/L +/-136.5U/L and 466.2 U/L +/-82.8 U/L, 732.9 U/L +/-91.1 U/L, respectively) were significantly greater than those in IP group. CONCLUSION: These results suggest that p44/42 MAPK pathway plays a vital role in the protection of hepatocytes in ischemic preconditioning.

Alanine Transaminase↗
PubMed1996

Anomalous diffusion of water in biological tissues.

This article deals with the characterization of biological tissues and their pathological alterations. For this purpose, diffusion is measured by NMR in the fringe field of a large superconductor with a field gradient of 50 T/m, which is rather homogenous and stable. It is due to the unprecedented properties of the gradient that we are able not only to determine the usual diffusion coefficient, but also to observe the pronounced Non-Debye feature of the relaxation function due to cellular structure. The dynamics of the probability density follow a stretched exponential or Kohlrausch-Williams-Watts function. In the long time limit the Fourier transform of the probability density follows a long-tail Lévy function, whose asymptotic is related to the fractal dimension of the underlying cellular structure. Some of the properties of Lévy walk statistics are discussed and its potential importance in understanding certain biophysical phenomena like diffusion processes in biological tissues are pointed out. We present and discuss for the first time NMR data giving evidence for Lévy processes that capture the essential features of the observed power law (scaling) dynamics of water diffusion in fresh tissue specimens: carcinomas, fibrous mastopathies, adipose and liver tissues.

Adipose Tissue↗
PubMed1986

[Changes in the anatomic structures and cellular composition of lymph nodes after prenatal exposure to oleandomycin].

Tracheobronchial and mesenteric lymph nodes have been investigated in 120 embryos, fetuses and newborn (5-, 14-, 30-day-old) rats by means of anatomical, histological and electron microscopical methods. Prenatal influence of oleandomycin phosphate on development of lymph nodes has been analysed. Effect of the drug on the mother--fetus system produces certain reactive changes in the anatomical structures responsible for the barrier-filtration and drainage functions of the lymph nodes. The immunocytopoietic function is inhibited, as demonstrates certain decrease of lymphatic line cells.

Animals↗
PubMed1996

[Characterization of structural and cellular components in pulmonary sarcoidosis granuloma].

Granulomas occurring in sarcoidosis with lung involvement are mostly located in the paravasal interstitium, pleura, bronchial mucosa and stroma. The phases and the activity of the disease process are characterised by different patterns from multicellular epitheloidcellular granulomas to marked hyalinisations and scarifications. For the purpose of histochemical characterisation of the composition of the cells and matrix of pulmonary granulomas in open and transbronchial lung biopsies of 15 patients suffering from sarcoidosis in different clinical stages, antibodies were employed against macrophages, neutrophil elastase, collagen types I and III, fibronectin, laminin, PCNA and against the tumour suppressor gene product P53. Identification was subsequently performed either by means of indirect immunofluorescence or the PAP technique. Multicellular granulomas showed, especially centrally, a specific fluorescence for macrophages involving also giant cells, whereas antibodies against neutrophil elastase could be mainly identified peripherally. PCNA and P53 protein were identified in the cytoplasm and partly also in the nuclei of giant cells. Collagen types I and III were mainly expressed pericentrally. Fibronectin was found in numerous multicellular epitheloid cellular granulomas not only in the peripheral collagen network but also centripetally oriented. The scarifying granulomas showed initially increased centripetal deposition of fibronectin followed by an addition of collagen types I and III. Laminin was always present in very small quantities only. The results obtained demonstrate a variable expression of matrix structures in sarcoidosis, dependent on the developmental stages of pulmonary granulomas, this expression being capable of control to some extent with the proportions of epitheloid cells, lymphocytes and macrophages that are present. Tumour suppressor gene p53 positive macrophage giant cells and adhesion molecules such as fibronectin participate in granuloma production to a varying extent.

Biopsy↗
PubMed1982

Developments in low-temperature biochemistry and biology.

Though under most circumstances harmful changes are induced in cellular structures by subzero temperatures, conditions can be found under which such damage is avoided. Thus, in solution, biochemical reactions can be slowed and more easily analysed and many enzyme-substrate complexes can be stabilized and structurally analysed; in crystals, 'stop-action' pictures unveil the stereochemical changes along reaction pathways. The progressive 'solidification' of non-covalent bonds involved in protein structures should permit investigation of their dynamics. Studies at high pressures open the way to new investigations on 'activated' enzyme-substrate complexes and might permit the refinement of current concepts to a considerable degree, as a preliminary but decisive step towards a full description of enzyme mechanisms. The conditions of medium allowing such cryobiochemical studies fail to protect cellular structures against cold. Investigations of plasma membrane behaviour are now under way to determine processes leading to cryosensitivity or cryotolerance of cells.

Animals↗
PubMed1999

Signal transduction networks and the biology of plant cells.

The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the completion of the genome sequence of Arabidopsis thaliana just around the corner, we can expect even faster progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are merging because signals pass between organelles and a single signal transduction pathway usually involves multiple organelles or cellular structures. Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure? This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of all these cellular structures need to be coordinated during ABA-induced stomatal closure. For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear envelope. This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile, the USA and the UK presented recent results from their laboratories.

Plant Cells↗
PubMed1993

Cryofixation and cryosubstitution: a useful alternative in the analyses of cellular fine structure.

A study of the fine structure of cultured mouse P815 cells as well as of mouse liver tissue after having undergone cryofixation and cryosubstitution is reported here. The P815 cells were cryofixed by a projection onto a liquid nitrogen (LN2), or liquid helium (LHe), cooled copper mirror: the liver tissue was processed (cryofixed) by high pressure freezing using a Balzers HPM 010 apparatus. No conventional chemical fixatives were used in the substitution medium which consisted of pure acetone. Embedding was carried out either in Lowicryl K11M, Lowicryl HM23, Epon or in LR White resins. The results of this study enabled us to conclude that a) one can obtain good preservation of cellular ultrastructure using different cryofixation methods and cryosubstitution without the use of chemical fixatives: b) different methods of embedding can be applied after various cryofixation techniques giving rise to slight differences in the well preserved fine structure; and c) high pressure freezing is to be recommended, in cryosubstitution studies, over that of slam freezing especially when cryofixing larger pieces of tissue which can result in a good morphology of up to 400 microns in depth.

Animals↗
PubMed1995

Structure and cellular physiology of Ca2+ stores in invertebrate photoreceptors.

Invertebrate microvillar photoreceptors contain an extensive, morphologically continuous endoplasmic reticulum (ER) that comprises several distinct subregions. Most prominent is the smooth submicrovillar ER, a sponge-like cisternal network underneath the photoreceptive microvillar membrane. The submicrovillar ER spatially separates the microvilli and a narrow space of submicrovillar cytoplasm from the remaining cell body, and, thus, defines a transduction compartment. In bee and locust photoreceptors, the shape and position of these submicrovillar ER cisternae is maintained by interaction with actin filaments. The structural layout of the ER is either rather static, or, in some invertebrate species, the ER undergoes dramatic rearrangements during illumination. The submicrovillar ER has a high Ca content in dark-adapted cells (47.5 mmol/kg dry weight in bee photoreceptors), and acts as a source and sink for Ca2+ mobilized by illumination. About 50% of the Ca content is released by a 3 s, non-saturating light stimulus, and an almost equimolar amount of Mg is taken up to maintain electroneutrality within the ER. Ca2+ release is initiated by Ins(1,4,5)P3. In addition, the submicrovillar ER contains a heparin-insensitive, caffeine- and ryanodine-sensitive Ca2+ release pathway in bee photoreceptors. Both the Ins(1,4,5)P3-dependent and the ryanodine-sensitive Ca2+ release mechanism are modulated by cytosolic Ca2+, but at different Ca2+ concentrations. The presence of two release pathways with different Ca2+ sensitivities may be a prerequisite for highly localized, exceptionally fast and large Ca2+ elevations during the illumination of invertebrate photoreceptors.

Animals↗