Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension

Mathew, Rajamma, Elizabeth S. Gloster, T. Sundararajan, Carl I. Thompson, Guillermo A. Zeballos, andMichael H. Gewitz. Role of inhibition of nitric oxide productionin monocrotaline-induced pulmonary hypertension. J. Appl. Physiol. 82(5): 1493-1498, 1997.Monocrotaline (MCT)-induced pulmonary hypertension (PH) isassociated with impaired endothelium-dependent nitric oxide(NO)-mediated relaxation. To examine the role of NO in PH,Sprague-Dawley rats were given a single subcutaneous injection ofnormal saline [control (C)], 80 mg/kg MCT, or the same doseof MCT and a continuous subcutaneous infusion of 2 mg · kg¹ · day¹of molsidomine, a NO prodrug (MCT+MD). Two weeks later, plasma NO₃ levels, pulmonary arterialpressure (Ppa), ratio of right-to-left ventricular weights (RV/LV) toassess right ventricular hypertrophy, and pulmonary histology wereevaluated. The plasma NO₃ level inthe MCT group was reduced to 9.2 ± 1.5 µM(n = 12) vs. C level of 17.7 ± 1.8 µM (n = 8; P < 0.02). In the MCT+MD group,plasma NO₃ level was 12.3 ± 2.0 µM (n = 8). Ppa and RV/LV in theMCT group were increased compared with C [Ppa, 34 ± 3.4 mmHg(n = 6) vs. 19 ± 0.8 mmHg(n = 8) and 0.41 ± 0.01 (n = 9) vs. 0.25 ± 0.008 (n = 8), respectively;P < 0.001]. In the MCT+MDgroup, Ppa and RV/LV were not different when compared with C [19 ± 0.5 mmHg (n = 5) and 0.27 ± 0.01 (n = 9), respectively;P < 0.001 vs. MCT]. Medial wall thickness of lung vessels in the MCT group was increased comparedwith C [31 ± 1.5% (n = 9)vs. 13 ± 0.66% (n = 9);P < 0.001], and MDpartially prevented MCT-induced pulmonary vascular remodeling [22 ± 1.2% (n = 11);P < 0.001 vs. MCT and C].These results indicate that a defect in the availability of bioactive NO may play an important role in the pathogenesis of MCT-induced PH.

     

FLORAL INITIATION AND EARLY DEVELOPMENT IN ERIGERON PHILADELPHICUS (ASTERACEAE)

The order of floral initiation and subsequent organogeny of Erigeron philadelphicus L. (Asteraceae: Astereae) was found to deviate from the acropetal pattern generally reported for the Asteraceae. Light micrographs show periclinal divisions in the first, second, and deeper subsurface layers of cells on the flanks of the inflorescence apex as the earliest evidence of floral initiation. Scanning electron microscope micrographs indicate that the disk flowers appear first and arise as small protuberances approximately one-third of the way up the previously and undifferentiated highly convex inflorescence apex. A succession of disk flowers arises acropetally in a complex anthotaxy characterized by about 21 dextrorse and 12–15 sinistrorse parastichies (although this pattern is obscured at the apex). After one to three disk flowers have been initiated in each parastichy, the first ray flower initials can be seen to initiate in sites proximal to the oldest and largest disk flowers. Additional ray flowers then initiate basipetally following the dextrorse parastichies established by the disk flowers. Overall floral initiation on the inflorescence apex proceeds acropetally for the disk flowers and basipetally for the ray flowers until the available space is filled. Floral development adheres to the same plan—proceeding bidirectionally on the inflorescence meristem with the oldest and most complete flowers of both types located on the equator established at initiation.     

Identification of the Axin and Frat binding region of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression.     

Short peptide receptor mimics for atherosclerosis risk assessment of LDL

Short peptides sequences were selected that showed binding selectivity towards healthy or oxidised (unhealthy) low density lipoprotein (LDL), respectively. These were investigated for application in atherosclerosis risk monitoring. Comparison was also made with the LDL receptor ligand repeat peptide (LR5). The peptides were immobilised on a gold surface plasmon resonance surface and LDL binding detected as a shift in the resonance. 3.7x10(7) (+/-5.6x10(6)) LDL/mm(2)/microg/ml solution LDL were bound on GlySerAspGlu-OH and 6.8x10(7) (+/-9.2x10(6)) LDL/mm(2)/microg/ml on GlyCystineSerAspGlu, compared with approximately 10(8) LDL/mm(2)/microg/ml on LR5. In this first group, binding of LDL decreased with oxidation level and a good correlation was found between LDL binding and residual amino groups on the apoprotein of the LDL following oxidation, or the change in relative electrophoretic mobility (REM) of LDL. The decrease in binding was 1.1x10(7) LDL particles/mm(2) per% oxidation for GlySerAspGlu-OH, 1.8x10(7) LDL particles/mm(2) per% oxidation for GlyCystineSerAspGlu and 2.4x10(7) LDL particles/mm(2) per% oxidation for LR5. A second group of three peptides were also selected showing increased binding with LDL oxidation: GlyCystineCysCys (1.5x10(7) LDL/mm(2) per microg/ml), GlyLysLysCys-SH (10(7) LDL/mm(2) per microg/ml) and GlyLysLys-OH (5.6x10(7) LDL/mm(2) per microg/ml). The latter gave a linear increase in LDL binding with oxidation level (1.2x10(7) LDL particles/mm(2) per% oxidation). LDL concentration is around 2-3 mg/ml in plasma compared with the low detection levels with this method (1-10 microg/ml), allowing a strategy to be developed requiring the minimum sample volume and diluting with physiological buffer prior to assay. By using a comparative reading between LDL adsorption on surfaces from the first and second group of peptides (e.g. GlyCystineSerAspGlu and GlyLysLys-OH, respectively), LDL oxidation could be determined without knowledge of LDL concentration. Higher binding was seen on GlyCystineSerAspGlu than GlyLysLys-OH below 30% LDL oxidation, whereas above 30% oxidation the binding on the latter surface was greater. Simple correlation of this form could provide good tests for atherosclerosis risk.     

A model of bacterial intestinal infections in Drosophila melanogaster

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.