Electron microscopic analysis of glucose-induced endothelial damage in primary culture: possible mechanism and prevention.

We previously reported that high glucose treated cultured endothelial cells (ECs) showed intercellular gaps by transmission electron microscopy (TEM). These gaps were abrogated with insulin and/or heparin treatment. Our aims were to assess the severity of injury in ECs treated with high glucose for variable duration, and to further study the protective effects of insulin and/or heparin. Cells were also treated with L-buthionine sulfoximine (BSO), a glutathione inhibitor, to help understand the mechanism of high glucose injury. Primary porcine ECs were treated with high glucose (30 mM) for 2, 6 or 10 days; and glucose plus insulin (1 U/ml), glucose plus heparin (5 microg/ml), glucose plus insulin plus heparin for 6 days. ECs were treated with BSO (0.001-0.05 mM) for 2 days. Pellets from trypsinized cells were processed for TEM. High glucose treatment revealed apoptosis or necrosis showing variable cell size, abnormal nuclei, condensation of nuclear chromatin, few mitochondria, cell membrane disruption and needle-shaped structures. Changes increased with duration of exposure. In high glucose plus heparin or insulin treated cultures at least one-half of the cells appeared normal. Most ECs were intact when treated with high glucose plus insulin plus heparin. BSO treatment showed dose-dependent changes with low doses showing apoptosis whereas higher doses revealed necrosis similar to high glucose treatment for 6 or 10 days. High glucose-induced EC injury increased with duration of exposure. These data demonstrate that high glucose injury resembles that of BSO treatment, suggesting that glutathione depletion may be involved in EC injury. Insulin and/or heparin protect against high glucose-induced injury.

The calculative nature of microbe-mineral interactions.

Microorganisms continually redefine themselves at many levels, including the molecule, cell, and community. Although it was initially assumed that this resulted from the genesis of information within DNA alone, it has since been shown that innovation originates at multiple levels. This occurs through calculative units, each unit consisting of two proliferating structures, one nested within the other and each undergoing changes in structural geometry that affect the proliferation rate of the other. For example, the recombination of genetic structures affects the proliferation of community structures, and the recombination of community structures affects the proliferation of genetic structures. The proliferation of a nested series of structures (e.g., genes proliferating within cells, cells proliferating within communities, communities proliferating within ecosystems) results in a logic circuit that calculates the form and function of each structural element in the series. In this situation each element functions as both a habitat and an inhabitant (environment and organism), and it is this dichotomy that determines the balance of nature. Nested geological structures, such as minerals and continents, also proliferate and redefine themselves in much the same way. Microbe-mineral interactions thus link nested biological calculations to an analogous set of nested geological calculations. Examples include the microorganisms involved in the nucleation (proliferation) of ferric hydroxides, carbonates, silicates, and ice crystals.

Localization of integrin alpha(v)beta3 and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in cutaneous and oral melanomas of dog.

Melanomas are common neoplasms of dogs and arise from pigment-producing cells called melanocytes or melanoblasts. Melanomas of skin are often easily cured by surgical excision, but those of oral mucosa are aggressive, metastasize to the regional lymph nodes and lungs, and respond poorly to conventional therapy. Tumor growth is sustained by proliferation of microvessels via a process called angiogenesis. Integrin alpha(v)beta3 is expressed in proliferating but not in quiescent microvessels suggesting a role in angiogenesis. Vascular endothelial growth factor (VEGF) manifests its mitogenic and angiogenic effects mainly via VEGF receptor-2 (VEGFR-2/Flk-1). We conducted this immunocytochemical study to investigate the expression of integrin alpha(v)beta3 and VEGFR-2 in archival and fresh samples from cases of canine melanomas. Results show that integrin alpha(v)beta3 was expressed in 72% and 88% of cutaneous and oral melanomas, respectively, and the expression was restricted to and immediately around the melanocytes and endothelial cells. VEGFR-2 staining of selected cases of melanoma revealed that its expression overlapped with the alpha(v)beta3 integrin. Dual immuno-gold electron microscopy confirmed co-localization of integrin alpha(v)beta3 and VEGFR-2 in melanocytes and endothelial cells. These data demonstrate expression and co-localization of integrin alpha(v)beta3 and VEGFR-2 in cutaneous and oral melanomas of dogs.

Development of a cell line from skin of goldfish, Carassius auratus, and effects of ascorbic acid on collagen deposition.

Growth characteristics and collagen expression were investigated in GFSk-S1, a cell line derived from the skin of an adult goldfish (Carassius auratus). These cells are anchorage dependent, grow well in Leibovitz-15 medium with 10% fetal bovine serum, and have been subcultured routinely for 5 years. Cells at various passages have been successfully cryopreserved and thawed. GFSk-S1 cells show mainly a fibroblastic morphology at low density, but at confluence islands of epithelial-shaped cells appear among the fibroblastic cells. The cells require little maintenance, and cultures have been kept viable for more than 3 months without medium changes. Although best growth was observed at room temperature, cell proliferation still occurred at 28 degrees C, and a subline was maintained and passaged for over a year at 25 degrees C. Cells were exposed to various concentrations of ascorbic acid, and its effects on collagen secretion were monitored by light and electron microscopy. Under phase-contrast microscopy, confluent GFSk-S1 cells exposed to ascorbic acid at 50 micrograms/ml showed distinct development of fibres as early as 3 days after treatment. Histochemical staining for collagen demonstrated a thick network of fibres under a monolayer of ascorbic acid-treated GFSk-S1 cells, and observation by transmission electron microscopy showed collagen fibres with typical banding pattern. This cell line appears to show a stable genotype, as collagen expression was induced at all passages. GFSk-S1 could be useful for studies not only of regulation of protein synthesis, but also of cell differentiation and wound healing.

Multicellular organization in a degradative biofilm community.

Diclofop methyl, a commercial herbicide, was used as the sole carbon source to cultivate diclofop-degrading biofilms in continuous-flow slide culture. The biofilms were analyzed by using scanning confocal laser microscopy and image analysis. Spatial relationships among members of the community were distinctive to diclofop-grown biofilms. These relationships did not develop when the biofilms were grown on more labile substrates but were conserved when the biofilms were cultivated with other chlorinated ring compounds. The structures included conical bacterial consortia rising to 30 mum above the surrounding biofilm, grape-like clusters of cocci embedded in a matrix of perpendicularly oriented bacilli, and other highly specific patterns of intra- and intergeneric cellular coaggregation and growth. These unique consortial relationships indicated that syntrophic interactions may be necessary for optimal degradation of diclofop methyl and other chlorinated ring compounds.

Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity.

A cell line, RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.

Co-existence of gametic and agametic seminiferous tubules in a chimeric mouse. A light- and electron-microscopic study.

Six chimeras, including 4 phenotypic males and 2 females, were produced by aggregation of F1 (C57BL x BALB/c) and Swiss white embryos. All were fertile, except 1 male, whose deviation in testicular structure prompted this light- and electron-microscopic study. This chimera had a well-developed sperm-conducting system, sperm in the epididymis and active accessory sex glands. The testes displayed typical parenchymal and stromal components with the important exception of co-existence of gametic and agametic seminiferous tubules. These tubules were organized in territories of quasi-lobular configurations which appeared to open separately into rete testis. The former corresponded to normally developed and active seminiferous tubules, while the latter were solid testicular cords devoid of any germ cells and embedded in solid masses of interstitial (Leydig) cells. Special mitochondrial transformations were identified in sustentacular (Sertoli) cells of both types of tubules, in maturing spermatids and sperm. These and other submicroscopic sperm defects might be the cause of infertility.

Ultrastructural study of synovial membrane from the antebrachiocarpal joint of calves.

The synovial intima from the antebrachiocarpal joint of 4-month-old calves was between 1 and 3 cells in thickness and did not have a basal lamina. Numerous areas of the intimal matrix were in direct contact with the joint lumen. The synovial membrane was comprised mainly of A-type synoviocytes usually located adjacent to the joint lumen. These cells were characterized by numerous filopodia (or lamellipodia), large, empty-appearing vacuoles, numerous lysosomes, large vacuoles containing granular material separated from the vacuolar membrane by a radiolucent band, and coated micropinocytotic vesicles. Smooth micropinocytotic vesicles were seen only rarely in these cells. In contrast, B-type cells had few filopodia, numerous smooth micropinocytotic vesicles, few coated micropinocytotic vesicles, a well-developed Golgi apparatus and rough endoplasmic reticulum, mitochondria that were longer and had a denser matrix than that of A cell mitochondria, and surprisingly, only few maturing or fully formed secretory granules. A distinct intermediate (C or AB) type synoviocyte could not be unequivocally identified. Desmosome-like structures were present between synoviocytes, although it was considered questionable if these were true intercellular junctions. No other junctions were present.

A Zoogloea sp. associated with blooms of Anabaena flos-aquae.

Bacteria were found attached to the heterocysts of Aphanizomenon flos-aquae and embedded within the mucilage of both anabaena flos-aquae and Microcystis aeruginosa in freshwater plankton. Electron microscopy of thin sections preceding the peak of an Anabaena flos-aquae bloom showed that the density of bacterial cells was 7.4 X 10(5) cells/ml in the planktonic macroenvironment and 2.6 X 10(11) cells/ml within the microenvironment of cyanobacterial mucilage. The bacteria occurred in aggregates and isolation required that these be dispersed by homogenizing at 50 000 rpm with glass beads. This procedure yielded a single bacterial isolate from blooms of Anabaena flos-aquae during 2 consecutive years. The isolate was flagellated, catalase- and oxidase-positive. Gram-negative, and rod-shaped to pleomorphic. Observation that the isolate required a pH greater than 8 for consistent growth, could not grow alone on liquid media but could grow alone on the corresponding solid media, could grow in liquid media only in the presence of Anabaena, formed tough mucilagenous colonies on solid media only in the presence of Anabaena extract, and rapidly assimilated but did not respire extracellular 14C-labelled organic matter produced by Anabaena suggested that the occurrence of the bacterium in cyanobacterial mucilage was not coincidental but reflected an obligatory bacterial requirement for the biological or physicochemical microenvironment of the mucilage. The bacterial isolate occurred in three growth forms. Either as a planktonic swarmer cell (which showed a positive chemotactic response to the cyanobacterium) embedded in cyanobacterial mucilage, or embedded in its own mucilage derived, in part, from a low molecular weight (below 1300) carbohydrate secreted by the cyanobacterium. These cultural, biochemical, and ecological characteristics suggest that the isolate is a new species in the genus Zoogloea and of potential importance in phytoplankton ecology.

Thermothrix thioparus gen. et sp. nov. a facultatively anaerobic facultative chemolithotroph living at neutral pH and high temperature.

Thermothrix thioparus gen. et ep. nov. occurs naturally in a New Mexico hot spring at a temperature of 74 degrees C, a pH of 7.0, and a HS- concentration of 1 mg/litre. The organism is gram-negative, non-motile, 0.5-1.0 X 3-20 mum, and forms cell chains up to 1 cm in length. The resulting filaments do not possess a sheath. Sulfur is deposited extracellularly. The organism was isolated using an autotrophic medium with HS- as the energy source and NO3- as the terminal electron acceptor. Anaerobically either NO2- or NO3- is required, NO2- is formed from NO3-, and no observable gas is evolved. Oxygen can also be used as the terminal electron acceptor, but growth is poor because of the decreased solubility of O2 at temperatures required for growth. Alternate energy sources used aerobically and anaerobically include hexose, HS-, SO3-, and S2O3=. The temperature optimum is 70-73 degrees C and growth occurs from 62 to 77 degrees C. The organism's thermal and physiological characteristics are compared to those of Bacillus stearothermophilus, Methanobacterium thermoautotrophicum, Sulfolobus acidocalderius, Thermus aquaticus, Thermus flavus, as well as Thiobacillus denitrificans, the latter being the only other facultatively anaerobic chemolithotroph which has been isolated and described.