Nitric oxide, a potent vasodilator of the aortic anastomosis in the estuarine crocodile, Crocodylus porosus

The effects of five neuropeptides (CGRP, SOM, SP, NPY, VIP), L-NAME (nitric oxide synthase inhibitor), and adrenaline on the contractile tone of the aortic anastomosis in the estuarine crocodile, Crocodylus porosus, were investigated. None of the neuropeptides, which had previously been found to be present in the aortic anastomosis, had any direct effect on the tension developed by ring preparations. L-NAME itself significantly increased the basal tone of the vascular ring preparations, suggesting a tonic release of nitric oxide in the preparation. Adrenaline produced concentration-dependent vasoconstrictions that were counteracted by profound reflex vasodilatations that were susceptible to blockade by L-NAME. Immunohistochemistry revealed the presence of nitric oxide synthase and tyrosine hydroxylase-containing (indicating the presence of a adrenergic innervation) nerve fibres in the adventitia and adventitio-medial border of the aortic anastomosis. These data demonstrate opposing actions of adrenaline and nitric oxide on the vascular smooth muscle in the anastomosis of the C. porosus. The morphology of the anastomosis, with the extremely thick muscular vessel wall, suggests a sphincter-like function for this vessel that could be controlled mainly by adrenergic and nitrergic mechanisms, (C) 2001 Academic Press.

Ripening and quality responses of avocado, custard apple, mango and papaya fruit to 1-methylcyclopropene

The potential for the ethylene binding inhibitor, 1-methylcyclopropene, to delay ripening of 'Hass' avocado, 'African Pride' custard apple, 'Kensington Pride' mango and 'Solo' papaya was examined. Fruit were gassed with 25 muL/L 1-methylcyclopropene for 14 h at 20 degreesC, followed by treatment with 100 muL/L ethylene for 24 h, and then ripened at 20 degreesC. Ethylene treatment alone generally halved the number of days for fruit to reach the ripe stage, compared with untreated fruit. 1-Methylcyclopropene treatment alone increased the number of days to ripening by 4.4 days (40% increase), 3.4 days (58%), 5.1 days (37%) and 15.6 days (325%) for avocado, custard apple, mango and papaya, respectively, compared with untreated fruit. Applying 1-methylcyclopropene to the fruit before ethylene prevented the accelerated ripening normally associated with ethylene treatment, so that the number of days to ripening for fruit treated with 1-methylcyclopropene plus ethylene was similar to the number of days to ripening for fruit treated with 1-methylcyclopropene alone. 1-Methylcyclopropene treatment was associated with slightly higher severity of external blemishes in papaya and custard apple, slightly higher rots severity in avocado, custard apple and papaya, and at least double the severity of stem rots in mango, relative to fruit not treated with 1-methylcyclopropene. Thus, 1-methylcyclopropene treatment has the potential to reduce the risk of premature ripening of avocado, custard apple, mango and papaya fruit due to accidental exposure to ethylene. However, additional precautions may be necessary to reduce disease severity associated with 1-methylcyclopropene treatment.

Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis

Background & Aims: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. Methods: Rats were treated with captopril (100 mg kg(-1) day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals sewed as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. Results: Cap topril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. Conclusions: Captopril significantly attenuates the progression of hepatic fibrosis in the vat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis. (C) 2001 Elsevier Science B.V. All rights reserved.

Hemoglobin-degrading, aspartic proteases of blood-feeding parasites - Substrate specificity revealed by homology models

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH2]Leu-Tyr-Tyr-Ser-NH2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current, study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways. Cell Motil. Cytoskeleton 49:93-103, 2001. (C) 2001 Wiley-Liss, Inc.

Two new classes of conopeptides inhibit the alpha 1-adrenoceptor and noradrenaline transporter

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha (1)-adrenoceptors (rho -TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi -MrIA and chi -MrIB from the mollusk-hunting C. marmoreus). rho -TIA and chi -MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Antagonism of the two-needle pine stem rust fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum in vitro and in planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degreesC. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wail and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host tell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta -1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Pharmacotherapy of intermittent claudication

Intermittent claudication (IC) is leg muscle pain, cramping and fatigue brought on by exercise and is the primary symptom of peripheral arterial disease. The goals of pharmacotherapy for IC are to increase the walking capacity/quality of life and to decrease rates of amputation. In 1988, pentoxifylline was the only drug that had reasonable supportive clinical trial evidence for being beneficial in IC. Since then a number of drugs have shown benefit or potential in IC. Cilostazol, a specific inhibitor of phosphodiesterase 3 and activator of lipoprotein lipase, clearly increases pain-free and absolute walking distances in claudicants. However, cilostazol does cause minor side effects including headache, diarrhoea, loose stools and flatulence. Naftidrofuryl, a serotonin (5-HT2) receptor antagonist and antiplatelet drug, is beneficial in claudicants. Inhibitors of platelet aggregation (including nitric oxide from L-arginine or glyceryl trinitrate) and anticoagulants (low molecular weight heparin, defibrotide) probably have both short and long-term benefits in IC. In addition, intravenous infusions of prostaglandins (PGs) PGE1 and PGI2 have an established role in severe peripheral arterial disease and the recent introduction of longer lasting and/or oral forms of the PGs makes them more likely to be useful in the IC associated with less severe forms of the disease. There are some exciting new approaches to the treatment of IC, including propionyl-L-carnitine and basic fibroblast growth factor (bFGF).

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

alpha-melanocyte stimulating hormone potentiates p16/CDKN2A expression in human skin after ultraviolet irradiation

The contribution of the UV component of sunlight to the development of skin cancer is widely acknowledged, although the molecular mechanisms that are disrupted by UV radiation (UVR) resulting in the loss of normal growth controls of the epidermal stem cell keratinocytes and melanocytes is still poorly understood. alpha-Melanocyte stimulating hormone (alpha-MSH), acting via its receptor MC1, has a key role in skin pigmentation and the melanizing response after exposure to UVR. The cell cycle inhibitor p16/CDKN2A also appears to have an important function in a cell cycle checkpoint response in skin after exposure to UVR. Both of these genes have been identified as risk factors in skin cancer, MC1R variants are associated with increased risk to both melanoma and nonmelanoma skin cancers, and p16/CDKN2A with increased risk of melanoma. Here we demonstrate that the increased expression of p16 after exposure to sub-erythemal doses of UVR is potentiated by alpha-MSH, a ligand for MC1R, and this effect is mimicked by cAMP, the intracellular mediator of alpha-MSH signaling via the MC1 receptor. This link between p16 and MC1R may provide a molecular basis for the increased skin cancer risk associated with MC1R polymorphisms.

A sponge allelochemical induces ascidian settlement but inhibits metamorphosis

Secondary metabolites synthesised by sessile invertebrates appear to play a role in creating and maintaining space on hard substrata by repelling competitors. In this study, we investigated the responses of the larvae of the ascidian Herdmania curvata to haliclonacyclamine A (HA), the major component of a suite of cytotoxic alkaloids extracted from the sponge Haliclona sp. 628. Both Haliclona sp. 628 and Herdmania curvata inhabit the crest and slope of Heron Island Reef. High rates of settlement were induced in competent H. curvata larvae by a range of concentrations of HA, all lower than that naturally occurring in the sponge. HA did not induce precompetent larvae to settle. Although early metamorphosis of HA-induced larvae was normal, larvae exposed to all but the lowest concentration of HA were developmentally arrested after completion of tail resorption, at about 4 h after the initiation of metamorphosis. These postlarvae underwent extensive cellular necrosis within 24 h. We also demonstrate that the addition of a transcriptional inhibitor, actinomycin D, to larvae also causes inhibition of metamorphosis after tail resorption is completed. Analyses of incorporation of radiolabelled nucleotides to measure levels of transcription during normal development and after the addition of the transcriptional inhibitor indicate that there is a significant burst of transcriptional activity just after tail resorption is completed. Despite inhibiting metamorphosis at the same stage as actinomycin D, HA increases initial rates of RNA synthesis after induction of metamorphosis in a manner similar to that observed in normal postlarvae until the onset of cellular necrosis. We conclude that HA initially induces H. curvata larvae to settle and progress through early metamorphosis possibly by engaging the same pathway as other artificial and environmental cues but subsequently inhibits completion of metamorphosis, resulting in death of the postlarvae. Since HA does not affect overall transcription rates, it appears to disrupt another important developmental process during early metamorphosis.

Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR) -containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor. using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general. cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, interior/exterior orientations of side chains. and backbone conformation of the LRR structures can be predicted correctly. On the other hand. the analysis revealed that, in some cases. it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization

Friedreich ataxia (FA) Is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (beta-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and beta-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis. (C) 2002 by The American Society of Hematology.