The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.     

Crystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residues

BACKGROUND AND PURPOSE: Early and accurate diagnosis of brain edema in stroke patients is essential for the selection of appropriate treatment. We examined the correlations between the changes in the apparent diffusion coefficient (ADC), regional water content, and tissue ultrastructure during early focal cerebral ischemia. METHODS: The left middle cerebral arteries of cats were occluded with an intramagnet occlusion/recirculation device. T2-weighted, diffusion-weighted, and perfusion imaging were performed repeatedly during the initial 3 hours after occlusion. The ADCs obtained from ADC maps were compared with the corresponding tissue water content values determined by gravimetry and electron microscopic water localization. RESULTS: ADC reduction was detected in areas of low perfusion 15 minutes after occlusion and thereafter. The water content increase correlated linearly with the ADC decreases in both the gray and white matter. However, both the water content corresponding to an ADC value and the rate of ADC change of the gray and white matter differed significantly (P<.05) as follows: y = -10105x + 8533 (r=.86) and y = -6174x + 4611 (r=.67), respectively, where x is the water content (grams water per gram tissue) and y is the ADC (x 10(-6) mm2/s). Hydropic astrocytic swelling was seen in both structures, and in the white matter, oligodendroglial and myelinated axonal swelling and periaxonal space enlargement were observed. CONCLUSIONS: When early ischemic edema in experimental focal cerebral ischemia is evaluated with ADC mapping, the different slopes and intercepts of the water content and ADC correlation lines for the gray and white matter, which probably reflect different ultrastructural localization of water, should be taken into account.     

Effect of intracolonic benzalkonium chloride on trinitrobenzene sulphonic acid-induced colitis in the rat

BACKGROUND: We investigated the effects of benzalkonium chloride (BAC) on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. METHODS: TNBS was administered intrarectally before and/or after BAC treatment. In the first study, the effects of treatment with BAC 6, 12 or 24 h after TNBS were examined. In the second study, animals were treated with BAC before, after or before and after TNBS, and were examined 7 days later. The severity of colitis was assessed by macroscopic and histological scoring of the colonic damage and by determination of colonic myeloperoxidase (MPO) activity. Macrophages and CD4+ and CD8+ T cells were examined by immunohistochemistry. RESULTS: When BAC was instilled into the colon 6, 12 or 24 h after TNBS, weight loss and macroscopic and histological features of the colon were similar to that of controls (TNBS alone). In contrast, MPO activity was significantly reduced in all three groups post-treated with BAC. In the groups examined 7 days after TNBS treatment, rats post-treated with BAC exhibited increased weight gain and significantly reduced macroscopic damage and MPO activity compared to the TNBS control group. Rats pre-treated with BAC exhibited less macroscopic damage of the colon than rats receiving only TNBS, but histological damage, MPO and weight gain were unchanged from TNBS controls. Immunohistochemistry revealed that BAC pre-treatment increased the numbers of macrophages and T cells in the colon. After TNBS treatment, macrophage accumulation was evident in the colon, but T cells were scarce. However, these cells were preserved or enhanced in the colonic mucosa in TNBS-treated rats that had been pre-treated with BAC. CONCLUSIONS: Treatment with BAC, particularly after induction of colitis, produces a significant reduction in the severity of tissue injury and inflammation through mechanisms that are not fully understood.     

Nonradioactive Northern blotting with biotinylated and digoxigenin-labeled RNA probes

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called Chem-Link, that quickly and conveniently labels RNA for use in Northern blotting.     

Fibronectin fragments modulate human retinal capillary cell proliferation and migration

Capillary morphogenesis involves cell-cell and cell-matrix interactions. Proteases elaborated by capillary cells modify the extracellular matrix (ECM) to facilitate capillary tube formation. Previously, we detected the presence of fibronectin fragments (Fn-f) associated with the proform of matrix metalloprotease-2 (MMP-2) in conditioned medium of human retinal endothelial cells (HRECs). Association of this fragment to latent MMP-2 prevented autocatalytic activation of MMP-2, suggesting a modulatory role of Fn-f in MMP-2 activation. In this report, we examined the potential role of Fn-f on two processes involved in angiogenesis, proliferation and migration of vascular cells. The effects of Fn-f on proliferation were determined by DNA synthesis and cell counts. Their effects on migration were assessed using modified Boyden chambers. Seven Fn-f were tested on vascular cell migration and/or proliferation. Three Fn-f induced migration. Fn-f of 30-kDa and 120-kDa size positively affected proliferation of microvascular cells but not macrovascular cells. A 45-kDa gelatin binding fragment of Fn inhibited HREC proliferation but stimulated pericyte and smooth muscle cell proliferation. The potency of these fragments exceeded that of the known angiogenic growth factor, basic fibroblast growth factor (bFGF), on HREC migration. ECM components such as fibronectin may influence capillary morphogenesis by the generation of fragments that can modulate proliferation, migration, and protease activation. In the setting of diabetes, excess Fn is generated and is available for degradation. Thus, the production of Fn-f may be specifically relevant to the angiogenesis observed in proliferative diabetic retinopathy.     

Hemodynamic characteristics, myocardial kinetics and microvascular rheology of FS-069, a second-generation echocardiographic contrast agent capable of producing myocardial opacification from a venous injection

OBJECTIVES: We sought to 1) study the effects of FS-069 on cardiac and systemic hemodynamic function, myocardial blood flow, left ventricular wall thickening and pulmonary gas exchange when injected intravenously; and 2) compare the myocardial kinetics and microvascular rheology of FS-069 and Albunex when injected directly into a coronary artery. BACKGROUND: FS-069 is a second-generation echocardiographic contrast agent composed of perfluoropropane-filled albumin microspheres; it is capable of consistent and reproducible myocardial opacification from a venous injection. METHODS: Nine dogs were used to study the effects of FS-069 on hemodynamic function, pulmonary gas exchange, left ventricular wall thickening and myocardial blood flow and to characterize its myocardial kinetics when injected intravenously. These dogs were also used to compare the myocardial kinetics of FS-069 with those of Albunex during intracoronary injections. Nine Sprague-Dawley rats were used to compare the microvascular rheology of these two contrast agents, and in vitro modeling was performed to assess whether the microvascular findings of FS-069 can explain its echocardiographic behavior during direct coronary injections. RESULTS: There were no effects of 30 rapid venous injections of FS-069 (every 20 s) on cardiac output; mean aortic, pulmonary or left atrial pressures; and peak positive and negative first derivative of left ventricular pressure (dP/dt). Similarly, there were no effects of this agent on radiolabeled microsphere-measured regional myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange. When injected intravenously, the myocardial transit of this agent resembled a gamma-variate form. When diluted FS-069 was injected directly into the coronary artery; however, its transit resembled the integral of gamma-variate function, with persistent myocardial opacification lasting several minutes, which was different from that of Albunex. Intravital microscopy revealed that, unlike Albunex, when no bubbles are entrapped within the microcirculation after an arterial injection, a very small fraction of the diluted, larger FS-069 microbubbles are entrapped. In vitro modeling confirmed that this small fraction of microbubbles can result in persistent myocardial opacification. CONCLUSIONS: FS-069 produces no changes in hemodynamic function, myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange when injected intravenously in large amounts. When diluted FS-069 is injected into the coronary artery, a very small fraction of the larger bubbles are entrapped within the microcirculation, resulting in a persistent contrast effect. Thus, although FS-069 is a safe intravenous echocardiographic contrast agent, it cannot provide information on myocardial blood flow when injected directly into a coronary artery.     

Comparison of ras-p21 bound to GDP and GTP: differences in protein and ligand dynamics

This paper documents the first essential dynamics analysis of ras protein ligands and of the protein itself, showing important features of their dynamic properties. Essential dynamics analysis of 300 ps of full solvent molecular dynamics simulations revealed differences in structure and dynamics between GDP- and GTP-bound forms of H-ras-p21. Regions in the protein which exhibited a structural shift correspond to the switch regions described previously. Differences in dynamics between H-ras-p21 GDP and H-ras-p21 GTP may be related to interactions of ras with GAP and its receptor and effector. Molecular dynamics of free GDP (in the absence of protein) were performed in water for 2 ns and analysed using essential dynamics. The conformations of GDP and GTP when bound to the protein were compared with free GDP, revealing that the ligands bind to the protein in an energetically unfavourable conformation. GDP and GTP molecules from various other protein crystal structures were also analysed. These ligands adopt similar conformations to those seen in H-ras-p21.      

Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria

Apoptotic cell death involves a ritual series of morphological changes, presumably reflecting a conserved molecular pathway. We now report that the nuclear events typical of apoptosis can be reproduced in "apoptotic" Xenopus egg extracts. In this cell-free system, nuclear assembly and protein import are initially normal; after 2-4 hr, however, a process of nuclear destruction ensues involving chromatin condensation and the shrinkage and fragmentation of the nuclei. This apoptotic process, which also occurs in nuclei added exogenously, is blocked by the addition of baculovirus-expressed Bcl-2 protein. To block the disintegration of nuclei that are added later, Bcl-2 must be present during this latent period. "Apoptosis" in these extracts requires a dense organelle fraction enriched in mitochondria. The cell-free system described here provides a novel tool for understanding intracellular events in apoptosis and the inhibitory function of Bcl-2.     

Biocontrol potential of the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae for tsetse flies (Glossina spp.) at developmental sites

Belonging to the Annonaceae family, marolo (Annona coriaceae) is a native species of the Brazilian "cerrado" región (Minas Gerais, Goiás and Distrito Federal) and can be found in South American tropical zones. Its fruits are highly consumed by local people and commercialized in markets or street stalls. There is, however, a tendency for the extinction of marolo due to deforestation and the large scale plantation of monocultures instead of native plants. The literature still offers no data on the chemical composition of the proximate composition and vitamin C, A and tannin contents were carried out on the yellow marolo pulp as well as the determination of the physico-chemical characteristics of the seed oil. Five batches of fruit from the Alfenas region--south of Minas Gerais State--were analysed in this work and their average composition were: humidity 77%, total sugar 15%, reducing sugar 11%, crude protein 1%, lipids 3%, fiber 5% and fixed mineral residue 1%. The contents of vitamin C and A were 8.2 mg/100g and 117.5 RE/100g, respectively, and the tannin content was 245 mg/100g. The results showed high fiber and lipid contents of marolo pulp in comparison with many other tropical fruit pulps. The vitamin C contents were equivalent to those found in avocado, pineapple and watermelon, while the vitamin A contents were equivalent to papaya, peach, guava and several other tropical fruits. Marolo seed contains 45% of oil on a dry basis. Its composition and physico-chemical characteristics showed the possibility of producing a good quality oil, with great potential for the fine oil market. However the presence of alkaloids in the oil needs to be further studied. Their elimination could be done by refining or extraction in a continuous press. The results exalt the high quality of marolo pulp, showing that the preservation of native species should be stimulated.