Intracellular calcite and sulfur dynamics of Achromatium cells observed in a lab-based enrichment and aerobic incubation experiment.

We investigated the intracellular dynamics of calcite and sulfur in the large sulfur-oxidizing, calcite-accumulating bacterium Achromatium, with an emphasis on oxygen exposure as a physiological control. For this purpose, morphological changes and possible accretion mechanisms of calcite granules in cells that were freshly collected from natural Achromatium-containing sediment were compared to cells from the same source after prolonged exposure to atmospheric oxygen. Intracellular sulfur is oxidized and removed in response to oxygen exposure. Calcite granules also undergo distinct oxygen-related dynamics; they alternate between tightly packaged, smooth granules with narrow but sharply defined interstitial spaces in atmospheric oxygen-exposed cells, and more loosely packaged granules with irregular, bumpy surface texture and larger interstitial spaces in cells that were not artificially exposed to oxygen. These results suggest that morphological changes of the calcite granules reflect their changing physiological role inside the cell. Sulfur oxidation and calcite dissolution appear to be linked in that proton generation during sulfur oxidation is buffered by gradual calcite erosion, visible in the smooth, rounded surface morphology observed after oxygen exposure. Our results support the hypothesis that calcite dynamics buffer the intracellular pH fluctuations linked to electron acceptor limitation during proton-consuming sulfide oxidation to sulfur, and electron acceptor abundance during proton-generating sulfur oxidation to sulfate.

Light microscope alignment methods.

Instructions for proper alignment for each of the most common wide field light microscopy contrast techniques are presented: bright field, polarization, dark field, phase contrast, differential interference contrast, and fluorescence. These are then followed by methods on photomicrography, measurement, and cleaning. An extensive set of Notes provides practical tips for each of these methods.

Immunohistochemical localization of matrixmetalloproteinase-2 in human coronal dentin.

UNLABELLED: While it is known that matrixmetalloproteinase-2 (MMP-2) is present in dentin, its distribution and role in human dentin formation and pathology are not well understood. OBJECTIVE: To characterize the distribution of MMP-2 in human coronal dentin. METHODS: Immunohistochemistry was used to investigate the distribution of MMP-2 in coronal dentin. Freshly extracted human premolars and third molars (age range 12-30) were fixed with formaldehyde, demineralized with 10% EDTA (pH 7.4) and embedded in paraffin. Serial sections were made and subjected to immunohistochemical analysis using a specific monoclonal anti-MMP-2 antibody. Immunoreactivity was visualized with 3,3'-diaminobenzidine substrate and observed under light microscopy. ImageJ software was used to calculate the relative amount/distribution of MMP-2. RESULTS: The analysis revealed immunoreactivity for MMP-2 throughout human coronal dentin. However, intense immunoreactivities were identified in a 90-200 microm zone adjacent to the pre-dentin as well as a 9-10 microm wide zone adjacent to the dentinoenamel junction (DEJ). CONCLUSION: MMP-2 has a specific distribution in human coronal dentin indicating it's involvement in extracellular matrix organization in predentin and the establishment of the DEJ.

Adenosine produces nitric oxide and prevents mitochondrial oxidant damage in rat cardiomyocytes.

OBJECTIVE: To examine if adenosine prevents oxidant-induced mitochondrial dysfunction by producing nitric oxide (NO) in cardiomyocytes. METHODS AND RESULTS: Adenosine significantly enhanced the fluorescence of DAF-FM, a dye specific for NO, implying that adenosine induces synthesis of NO. Adenosine-induced NO production was blocked by both the nonspecific NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) and N(5)-(1-Iminoethyl)-l-ornithine dihydrochloride (l-NIO), an inhibitor of endothelial NOS (eNOS), but not by N(6)-(1-Iminoethyl)-l-lysine hydrochloride (l-NIL), an inhibitor of inducible NOS (iNOS), indicating that adenosine activates eNOS. Adenosine also enhances eNOS phosphorylation and its activity. The adenosine A(2) receptor antagonist 8-(3-chlorostyryl)caffeine but not the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the increase in NO production. CGS21680, an adenosine A(2) receptor agonist, markedly increased NO, further supporting the involvement of A(2) receptors. Adenosine-induced NO production was blocked by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2), a selective Src tyrosine kinase inhibitor, suggesting that Src tyrosine kinase is crucial for adenosine-induced NO production. Adenosine-induced NO production was partially reversed by both wortmannin and Akt inhibitor indicating an involvement of PI3-kinase/Akt. Pretreatment of cells with adenosine prevented H(2)O(2)-induced depolarization of mitochondrial membrane potential (DeltaPsi(m)). The protective effect was blocked by l-NAME and l-NIO but not by l-NIL, indicating that eNOS plays a role in the action of adenosine. The protective effect of adenosine was further suppressed by KT5823, a specific inhibitor of protein kinase G (PKG), indicating the PKG may serve as a downstream target of adenosine. CONCLUSION: Adenosine protects mitochondria from oxidant damage through a pathway involving A(2) receptors, eNOS, NO, PI3-kinase/Akt, and Src tyrosine kinase.

Mouse NG2+ oligodendrocyte precursors express mRNA for proteolipid protein but not its DM-20 variant: a study of laser microdissection-captured NG2+ cells.

Despite recent advances in our understanding of lineage of oligodendrocytes, detailed molecular characterization of this lineage in vivo is limited, primarily because of our inability to obtain a pure population of cells in situ. To define the molecular characteristics of oligodendrocyte lineage cells during development and their response to injury, we developed a strategy that uses laser capture microdissection (LCM) to isolate cells from sections and reverse transcription-PCR to determine mRNA expression. As a first step, we examined the expression of myelin-specific protein genes in NG2+ cells in cerebral cortex. We demonstrate that NG2+ cells in both developing and adult mice express NG2 mRNA but not mRNA for proteins specific for astrocytes, neurons, or microglia, indicating that a highly pure population of antigen-specific cells of the oligodendrocyte lineage can be obtained using LCM. Furthermore, we show that NG2+ cells express mRNAs for proteolipid protein (PLP), myelin basic protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase, but they dot not express DM-20 mRNA, a PLP mRNA splicing variant. Our data demonstrate that antigen-specific cells of oligodendrocyte lineage differentially express mRNA for myelin-specific proteins and their variants in vivo, partly define the gene expression in NG2+ cells, and raise questions about the cellular sites of DM-20 expression. This work also shows that LCM is a valuable tool to define and analyze gene expression in the cells of the oligodendrocyte lineage.

Temporal and morphologic characteristics of pinopod expression across the secretory phase of the endometrial cycle in normally cycling women with proven fertility.

OBJECTIVE: To assess the temporal and morphologic characteristics of pinopod expression on the surface of endometrium across the secretory phase, in LH-timed endometrial samples in normal, healthy women. DESIGN: Prospective, randomized study. SETTING: Academic teaching hospital. PATIENT(S): Sixty-eight healthy volunteers with proven fertility. INTERVENTION(S): Urinary LH-timed endometrial and blood sampling was performed on each subject on a randomly selected day of the secretory phase. MAIN OUTCOME MEASURE(S): Histologic dating, assessment of pinopods using scanning electron microscopy, and comparison with serum P levels. RESULT(S): Eighty-six endometrial tissue samples obtained from 68 subjects were evaluated under scanning electron microscopy. Pinopods were first observed on luteal day 5, corresponding with the onset of the midluteal phase increase in serum P levels. Pinopods persisted for the entire duration of the secretory phase, but their morphology changed as the cycle advanced. CONCLUSION(S): The present findings demonstrate that pinopods are a characteristic feature of the mid to late secretory phase endometrial epithelium, exhibit cycle-dependent changes in morphology, and are most prominent during the putative implantation interval.