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At least 1,279 records · Page 71Linked to original sources
PubMed1997

Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS.

In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied, following direct injection in several regions of the central and peripheral nervous system. The distribution of the cells expressing the transgene appears to be determined by natural anatomical boundaries and possibly by the degree of myelinization of a particular brain region. In highly myelinated areas with a compact cellular structure (e.g. the cortex and olfactory bulb) the spread of the viral vector is limited to the region close to the injection needle, while in areas with a laminar structure (e.g. the hippocampus and the eye) more widespread transgene expression is observed. Retrograde transport of the viral vector may serve as an attractive alternative route of transgene delivery. A time course of expression of beta-galactosidase in neural cells in the facial nucleus revealed high expression during the first week after AdLacZ injection. However, a significant decline in transgene expression during the second and third week was observed. This may be caused by an immune response against the transduced cells or by silencing of the cytomegalovirus promoter used to drive transgene expression. Taken together, the data underscore that for each application of adenoviral vectors as gene transfer agents in the nervous system it is important to examine vector spread in and infectability of the neural structure that is subject to genetic modification.

Adenoviruses, Human↗
PubMed1998

DNA topoisomerases: a new twist for antiparasitic chemotherapy?

The parasitic protozoa are notorious for their bizarre cellular structures and metabolic pathways, a characteristic also true for their nucleic acids. Despite these florid differences from mammalian cells, however, it has proven surprisingly difficult to devise novel chemotherapy against these pathogens. In recent years, the DNA topoisomerases from parasites have been the focus of considerable study, not only because they are intrinsically interesting, but also because they may provide a target for much-needed new antiparasitic chemotherapy.

Animals↗
PubMed2003

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR.

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

Bacillus cereus↗
PubMed2001

The Virtual Cell: a software environment for computational cell biology.

The newly emerging field of computational cell biology requires software tools that address the needs of a broad community of scientists. Cell biological processes are controlled by an interacting set of biochemical and electrophysiological events that are distributed within complex cellular structures. Computational modeling is familiar to researchers in fields such as molecular structure, neurobiology and metabolic pathway engineering, and is rapidly emerging in the area of gene expression. Although some of these established modeling approaches can be adapted to address problems of interest to cell biologists, relatively few software development efforts have been directed at the field as a whole. The Virtual Cell is a computational environment designed for cell biologists as well as for mathematical biologists and bioengineers. It serves to aid the construction of cell biological models and the generation of simulations from them. The system enables the formulation of both compartmental and spatial models, the latter with either idealized or experimentally derived geometries of one, two or three dimensions.

Biology↗
PubMed2001

Mechanism of the ultrasonic activation of micellar drug delivery.

The mechanism of the ultrasonic enhancement of the uptake of cytotoxic drugs, doxorubicin (DOX) and ruboxyl (Rb) by HL-60 cells from Pluronic micelles was studied. DOX and Rb sorption from either PBS or micellar Pluronic solutions is described by Langmuir-type isotherms characteristic of substrates with limited number of sorption centers. The sorption limits for Rb from PBS and Pluronic were considerably higher than those for DOX, presumably due to much higher Rb partitioning into cell membranes. The overall number of drug sorption centers for both drugs decreased in the presence of Pluronic implying the effect of Pluronic on the DNA conformation, which was confirmed by the electron paramagnetic resonance (EPR) experiments using Rb as a spin probe. Ultrasound increased drug uptake by the cells from PBS and Pluronic solutions. The fluorescence microscopy and flow cytometry experiments using fluorescently-labeled Pluronic showed that ultrasound enhanced both the intracellular uptake of Pluronic micelles and Pluronic trafficking into cell nuclei. A scheme is suggested that describes various equilibria controlling drug/cell interactions and effect of ultrasound on these equilibria. Under the action of ultrasound, the equilibrium between the micellar-encapsulated and free drug is shifted in the direction of free drug due to micelle perturbation; the equilibrium between extracellular and internalized drug is shifted to the intracellular drug due to the ultrasound-induced cellular changes that enhance the accessibility of various cellular structures to drug. An important advantage offered by ultrasound is that the same degree of the intracellular drug uptake may be achieved at a substantially lower drug concentration in the incubation medium.

Algorithms↗
PubMed1985

Acquired (digital) fibrokeratoma.

Histologic examination of fifty cases of acquired digital fibrokeratoma discloses three histologic variants of these lesions, viz: a tumor composed of thick dense and closely packed collagen bundles, a variant with an increased number of fibroblasts in the cutis, and a type with an edematous and poorly cellular structure. The acquired digital fibrokeratoma is considered as resulting from a neoformation of collagen by the fibroblasts. The acanthosis of the epidermis is probably secondary to the dermal alteration.

Adolescent↗
PubMed2002

Influence of tissue fixation on the binding of (125)I-angiotensin receptor ligands in the rat, mouse and rabbit kidney.

Aldehyde fixatives are often used to preserve tissue morphology and thereby aid in the identification of cellular structures expressing a target of interest. However, the effect of fixatives on target detection methods is unpredictable and it is currently unknown whether tissue fixation would allow the accurate detection of angiotensin AT(4) receptors in the kidney. In vitro receptor autoradiography on tissues fixed with 4% paraformaldehyde and 0.5% glutaraldehyde (+/-20% sucrose) had differing effects on the density of (125)I-AT(4) receptor ligand binding without affecting the tissue distribution of ligand binding in the rat and mouse kidney, whereas an increased expression of specific (125)I-AT(4) receptor ligand binding was found in the medulla region of the rabbit kidney. In contrast, such tissue fixation conditions dramatically decreased the renal binding of (125)I-angiotensin II receptor ligands, and altered the distribution of such ligand binding, in all three species. These results suggest that the method of tissue fixation and processing should be used cautiously in angiotensin receptor density measurements but can provide an accurate representation of kidney AT(4) receptor distribution only in the rat and mouse.

Angiotensin II↗
PubMed1988

An improved method for preserving the cellular membrane ultrastructure of in vitro cultivated cells using tannic acid.

Treatment of cultured cells with tannic acid after fixation with glutaraldehyde and osmification prevents the ultrastructural deterioration of cellular structures during dehydration and impregnation. Membranes of tannic acid-treated cells showed an increased contrast and sharp delineation of the polar zone. These results are discussed on the basis of simultaneous interactions of tannic acid with saturated phospholipids and OSO4. No ultrastructural differences are found between cells treated with tannic acids of different molecular weights.

Animals↗
PubMed2003

Ultrastructure of auxiliary and gonimoblast cells during carposporophyte development in the red alga Cryptopleura ruprechtiana (Delesseriaceae, Ceramiales, Rhodophyta).

The ultrastructure sequence for the complete post-fertilization development is described in Cryptopleura ruprechtiana (C. Agardh) Kylin, a member of the Delesseriaceae. Following fertilization the diploid nucleus is transferred to the auxiliary cell. This contains typical red algal proplastids, cytoplasmic concentric membranes, numerous small vacuoles and lipid bodies. Crystalline inclusions and virus-like particles are also present. In addition darkly staining spherical masses possibly represent dehydrated haploid chromatin. The multinucleate auxiliary cell produces initially one large gonimoblast initial and subsequently many smaller gonimoblast initials. The first formed generative gonimoblast cell is similar in cellular structure to the auxiliary cell. Gonimoblast initials are uninucleate but through caryokinesis they become multinucleate. They undergo repeated cleavage to form more gonimoblast cells. Subsequent, centripetal cytokinesis results in the formation of clusters of gonimoblast cells. A new type structural cap or association is observed in the septal plugs that interconnect gonimoblast initials. Terminal or generative gonimoblast cells cleave to form additional gonimoblast cells. Only terminal gonimoblast cells are differentiated to carpospores.

Animals↗
PubMed1997

Regulation of body water balance in reedfrogs (superspecies Hyperolius viridiflavus and Hyperolius marmoratus: Amphibia, Anura, Hyperoliidae) living in unpredictably varying savannah environments.

The regulation of body water balance was examined in the reedfrogs Hyperolius marmoratus taeniatus and Hyperolius viridiflavus ommatostictus. Temperature and stage of post-metamorphic development significantly affected the rate of water uptake. Hydrated reedfrogs prevented hyperhydration by voiding diluted urine when obtaining water. Within 48 hr after rehydration, body fluid osmolality remained at low levels, which may be supportive to counter excessive cutaneous water influx in hydrated frogs. Once evaporative water loss exceeded 10-12% total body mass, reedfrogs became anuric. The rate of water uptake strongly increased with increasing body water deficit. Both the anuric response and the increased rate of water uptake are assumed to strongly enhance the efficacy of using very briefly available water sources during dry-period conditions. Dry-adapted and estivating reedfrogs survived evaporative water losses between 40 and 55% total body mass. Bladder fluid stores contributed substantially to this desiccation tolerance. During a 16-day period of desiccation, H. v. ommatostictus could replace approximately 25% of evaporative water loss from the bladder fluid store. During desiccation, the level of free amino acids selectively increased in the gastrocnemius muscle tissue, which may support cell volume regulation and/or protect cellular structures from osmotic stresses. Even strongly dehydrated reedfrogs rehydrated quickly with no obvious osmoregulatory problem. Rehydration was associated with a higher than expected decrease of free amino acids in the gastrocnemius muscle tissue, a response that may help to protect cells from bursting during fast rehydration.

Amino Acids↗
PubMed1975

The basis, technique and indication for cryosurgery in tumours of the oral cavity and face.

The fundamentals of clinical application of crysurgery with very deep freezing of tissues is based on the necrotising effect of extreme cooling. The freezing of extracellular and intracellular free fluid leads to irreversible cell damage, because of a shift of electrolyte concentration to toxic values, and a direct lesion of internal cellular structures. The lesion due to cold occurs as a clear demarcation from the healthy surrounding, almost completely painlessness, and rapid healing without complication. For clinical application in the head and neck, there are cooling instruments fitted with specially shaped, exchangeable probe heads. Convincing results were achieved in the cryosurgical treatment of leucoplakias and Bowen's disease of the oral mucosa. The curative possibilities in carcinoma of the oral mucosa is small because of the limited performance of the cryo-carrier, which is best suited for superficial, non-infiltrating tumours. Preoperative rapid cooling of melanomas was performed to avoid dissemination of tumour cells during resection. Favourable functional results were achieved in the treatment of hemangiomas.

Bowen's Disease↗
PubMed1997

The organisations of hereditary information.

The meaning of hereditary information is not simple. It includes not only what a system receives and transmits but particularly what it makes. The syntactic basis to hereditary information is also not straightforward. For example, is DNA instructions, or data, or both? The answer to this question requires an appreciation of the meaning of the information yet there are a number of possible semantic systems for describing hereditary information including proteins and development. The descriptive boundaries of hereditary information are examined by locating some general organising themes including hierarchy, ecology, regulation, epigenetic systems and talkback. Though metaphors have limits in terms of their explanatory power a number have influenced the development of biological thinking and biosystems have variously been represented as chemical laboratories, computers, electromechanical machines and societies. In this article a further metaphor is discussed, that of life-as-a-play or dance in which the trio of script (genome), cast (metabolism) and stage (cellular structure) co-exist and pre-exist the phenotypic life history which inherits them. A fuller examination of this trio provides an important perspective on the study of the organisations of information processing in hereditary systems.

Genetic Code↗
PubMed1989

Sedimentation properties of chitosomal chitin synthetase from the wild-type strain and the 'slime' variant of Neurospora crassa.

Marked differences in the pattern of sedimentation of cellular structures were observed after isopycnic centrifugation of crude cell-free preparations from the Neurospora crassa wall-less 'slime' variant and mycelial wild-type strain. Kinetic studies of particle sedimentation showed that the various types of subcellular components, as revealed by turbidity, UV absorption, polypeptide patterns, and chitin synthetase activity determinations, sediment independently of one another. An important feature was the finding that chitin synthetase from 'slime' peaked at a median specific gravity of 1.1201 +/- 0.0036, whereas that from wild-type strain sedimented at a higher buoyant density (specific gravity 1.1349 +/- 0.0024). Different cultivation conditions or cell breakage procedures (osmotic lysis or ballistic disruption) did not seem to affect this sedimentation behavior. Electron microscopy revealed the presence of chitosomes (microvesicles containing chitin synthetase) in the chitin synthetase activity peaks obtained after isopycnic centrifugation of cell-free extracts from 'slime' and wild-type strains. The discrepancy in buoyant density of chitin synthetases from both N. crassa strains might point to inherent differences in chemical composition of the chitosomal microvesicles. In any case, the lower buoyant density of 'slime' chitosomes appears to be one of several major alterations in sedimentation behavior of subcellular structures. These alterations might be related to the inability of 'slime' to make a cell wall.

Centrifugation, Density Gradient↗
PubMed2003

Aberrant protein expression in cerebral cortex of fetus with Down syndrome.

Down syndrome is the most common birth defect associated with mental retardation. Identifying proteins that are aberrantly expressed therefore helps to understand how chromosomal imbalance leads to subnormal intelligence in Down syndrome. In the present study, we generated a fetal brain map with the use of an analytical method based on two-dimensional electrophoresis coupled with mass spectrometry and searched the proteome for differential protein expression. Among 49 proteins analyzed in seven control and nine Down syndrome fetuses, we found 11 proteins that have been deregulated in cerebral cortex of fetal Down syndrome. While double-strand break repair protein rad 21 homologue, eukaryotic translation initiation factor 3 subunit 5, mixed lineage leukemia septin-like fusion protein-B and heat shock protein 75 were increased; beta-amyloid precursor-like protein 1, tropomyosin 4-anaplastic lymphoma kinase fusion oncoprotein type 2, Nck adaptor protein 2, Src homology domain growth factor receptor bound 2-like endophilin B2, beta tubulin, septin 7 and hematopoietic stem/progenitor cells 140 were decreased. The current data suggest that misexpression of proteins that have functions ranging from signaling to cellular structural organization could contribute to or reflect brain dysgenesis in Down syndrome.

Case-Control Studies↗
PubMed1997

The cellular distribution of the adenomatous polyposis coli tumour suppressor protein in neuroblastoma cells is regulated by microtubule dynamics.

The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein. Using immunochemical techniques, this report corrects this observation. Immunoreactive bands of a size consistent with that of the full-length protein were observed by western blotting. Using immunocytochemistry, punctate immunoreactivity localized to areas of the cell containing microtubules, particularly neurite growth cones, in a distribution suggesting a role in neuritogenesis and growth cone extension. The protein did not localize to actin-rich cellular structures, and perturbation of the actin cytoskeleton had no effect upon this distribution. Treatment of cells with taxol to stabilize microtubules caused the concentration of the immunoreactive puncta to the tips of microtubules and areas along the axis of potential microtubule assembly. Treatment of cells with the microtubule disrupting reagent nocodazole showed that over shorter times the punctate distribution was not dependent upon polymerized microtubules. However, at longer incubation times a decrease in punctate immunostaining was observed. These results indicate that the intracellular distribution of the adenomatous polyposis coli protein is dependent upon microtubule but not actin dynamics. A role for this protein in the regulation of directed microtubule assembly is suggested.

Adenomatous Polyposis Coli↗
PubMed1985

What's new in light and electron microscopic immunocytochemistry? Application of the protein A-gold technique to routinely processed tissue.

The protein A-gold technique, a simple and reliable two-step postembedding immunocytochemical method, allows the light and electron microscopic detection of antigens in routinely fixed and embedded tissue. At the light microscopic level a permanent, nonfading stain is obtained. High resolution studies on intracellular antigens can be performed since the particulate nature of the colloidal gold permits accurate determination of the labeled cellular structures. The applicability of the protein A-gold technique for the localization of various peptide and protein antigens by light and electron microscopy is demonstrated.

Adenoma↗
PubMed1987

Intermediate filaments of myofibroblasts. Immunochemical and immunocytochemical analyses.

We generated a monoclonal anti-vimentin antibody, VIM-1, by mouse hybridoma technique, using an established myofibroblast line as a whole cell immunogen. The presence of vimentin polypeptides in the cultured myofibroblasts was confirmed by SDS-polyacrylamide gel electrophoresis and immunoblotting. By light microscopic immunocytochemistry, myofibroblasts in cultures as well as in frozen tissue sections showed a strong reaction with the anti-vimentin antibody, whereas these cells lacked either detectable desmin or cytokeratin. Our results support the fibroblastic origin of myofibroblasts. Immunoelectron microscopic study with ferritin-ABC technique demonstrated that VIM-1 reacted exclusively with 10-nm intermediate filaments, while other cellular structures revealed uniformly negative reaction against the antibody.

Antibodies, Monoclonal↗
PubMed2001

Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season.

The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test. In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%, respectively) were higher than during the breeding season in May (59 and 65.9%; P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%), suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors.

Acrosome↗