19-Hydroxyeicosatetraenoic acid analogs: Antagonism of 20-hydroxyeicosatetraenoic acid-induced vascular sensitization and hypertension

19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension.     

Tagging-via-substrate strategy for probing O-GlcNAc modified proteins

Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic analysis of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, we report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves three steps: metabolic labeling of cells with an unnatural GlcNAc analogue, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purification of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymatic systems among these cells from diverse biological species. We isolated azido-GlcNAc modified proteins from the cytosolic extract of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic analysis of O-GlcNAc modified proteins.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

The effect of morphactin on cauliflower curd-formation

Cauliflower plants in the juvenile phase treated on the leaf surface and at the apex with Morphactin solution (100 mg 1^-1) and this concentration failed to develop a curd and also to come to flowering. Instead of a curd the axis formed a conical or dome-shaped structure bearing ‘nodal’ rings. The number of rings increased with growth of the axis which either formed or did not form a small curd-like structure, which generally dried without flower formation.     

Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.     

Interspecific variation of body shape and sexual dimorphism in three coexisting species of the genus <Emphasis Type="Italic">Petrotilapia</Emphasis> (Teleostei: Cichlidae) from Lake Malawi

Differences in color patterns have been the most used feature in describing cichlid species belonging to genus Petrotilapia from Lake Malawi. In this study, we quantified morphological variation in body shape within and among three coexisting Petrotilapia species using landmark-based geometric morphometric methods. Statistic analyses revealed significant body shape differences among species but not between sexes. Post hoc multiple comparisons based on Mahalanobis distances revealed that P. nigra was significantly different from P. genalutea and Petrotilapia sp., whereas the latter two were not significantly different. The splines generated showed that the most pronounced variation was in the head region, in which P. nigra had a relatively longer and deeper head than the other two. The most clear-cut distinction was in gape length; P. genalutea had the longest gape, followed by Petrotilapia sp., whereas P. nigra had the shortest gape. Body depth was shallower in P. nigra than the others. When comparing sexes by their centroid size, ANOVA revealed that males were bigger than females. Therefore, we conclude that color is not the only feature that can distinguish these congeners. We discuss the observed sexual dimorphism in terms of sexual selection and relate morphological variation among species to feeding behavior, which may help explain their coexistence in nature.     

Trehalose Increases Freeze-Thaw Damage in Liposomes Containing Chloroplast Glycolipids

Chloroplast thylakoids contain three classes of glycolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). We have investigated the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC) and different chloroplast glycolipids during freezing to -18 degreesC, as a function of the presence of three sugars: glucose, sucrose, or trehalose. Contrary to the situation in thylakoids, where cryoprotection increases from glucose < sucrose < trehalose, liposomes containing 50% DGDG showed the opposite behavior. In fact, carboxyfluorescein leakage increased over the control values (freezing in the absence of sugar) in the presence of trehalose. This effect was not seen in vesicles made from pure EPC, or a mixture of EPC and MGDG, or EPC and SQDG. Liposomes made from mixtures of all three glycolipids, however, showed even more leakage in the presence of trehalose than liposomes containing only DGDG and EPC. Copyright 1998 Academic Press.