Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-I, tumor necrosis factor (TNF)-, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-I, TNF-, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5'-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5'-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC. vascular injury; nitric oxide; inflammation     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures

Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10)- and α-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.     

The crystal structure of JNK2 reveals conformational flexibility in the MAP kinase insert and indicates its involvement in the regulation of catalytic activity

c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called “MAP kinase insert”, which, for ERK2, has been shown to interact with regulatory proteins and, for p38α, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners.     

Fast drops: a high-throughput approach for setting up protein crystal screens.

There is significant demand to rapidly obtain protein structure information for both structural genomics and drug discovery applications. To meet this demand, all steps in the process of determining protein structure by X-ray crystallography need to be optimized and streamlined with high-throughput methodologies. This communication describes a method that brings high-throughput technology to protein crystallization in both manual and automated modes, suitable for virtually every crystallography laboratory.     

Plants used for reproductive health in Oaxaca,Mexico

Despite the existence of 2 government health centers in Mexico's San Francisco township, the use of herbal remedies for the treatment of reproductive health problems and the management of reproduction is almost universal. An ethnographic study conducted in 1980-1981 of 180 women and 126 women investigated use of specific herbal remedies in this highland area. The largest number of reproduction-related medicinal plants used San Francisco are for postpartum recovery. In fact, 80% of the women interviewed reported using only herbal remedies after their last delivery. These remedies are relied on to restore lost blood, heal the birth scar, and stop bleeding. Of the 40% of respondents who reported postpartum hemorrhaging, 70% used herbal remedies. Several of the plants used to treat menorrhagia and hemorrhaging are also used for infertility. The infertility remedies are believed to cleanse the blood and uterus, heal or strengthen the back so the fetus can adhere, help the womb retain the fetus, and cool the blood. Both infertility and miscarriage are attributed to incomplete postpartum recovery. Another large group of medicinal remedies is employed to speed labor or ease labor pains. These remedies are thought to stimulate blood flow or uterine contractions. An additional 15 plants are used to prevent or terminate pregnancy. The mechanism of action in these case is believed to be to both warm the blood to facilitate its flow and irritate the uterus so it will evacuate its contents. Reliance on these remedies derives from Chinantec ethnomedical understandings. Given the persistence of use of herbal remedies in many communities in the Third World, research is needed on their safety and efficacy.     

Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism

Exogenous nitric oxide (NO) triggers a preconditioning-like effect in heart via a pathway that is dependent on reactive oxygen species. This study examined the signaling pathway by which the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 2 microM) triggers its anti-infarct effect. Isolated rabbit hearts experienced 30 min of regional ischemia and 120 min of subsequent reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. Infarct size was reduced from 30.5 +/- 3.0% of the risk zone in control hearts to 10.2 +/- 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (2 microM) or the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (200 microM) completely blocked the infarct-sparing effect of SNAP (34.3 +/- 3.8 and 32.2 +/- 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (10 microM), which is a cell-permeable cGMP analog that activates protein kinase G, mimicked the preconditioning effect of SNAP by reducing infarct size to 7.5 +/- 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) or 5-hydroxydecanoate (100 microM; 28.9 +/- 2.7 and 33.6 +/- 5.0% infarction of the risk zone, respectively). To confirm these functional data and the effect of SNAP on the guanylyl cyclase-protein kinase G signaling pathway, cGMP levels were measured. SNAP increased the level from 0.18 +/- 0.04 to 0.61 +/- 0.14 pmol/mg of protein (P < 0.05). These data suggest that exogenous NO triggers the preconditioning effect by initiating a cascade of events including stimulation of guanylyl cyclase to make cGMP, activation of protein kinase G, opening of mitoK(ATP) channels, and, finally, production of reactive oxygen species.     

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.