Human melanoma cells express functional endothelin-1 receptors

Current evidence suggests that endothelium-derived factors enhance human melanoma vascular invasion. Therefore, we studied human melanoma cell expression of receptors to the endothelium-derived peptide, endothelin-1 (ET-1), and determined if they respond to ET-1 with proliferation and chemokinesis. Human metastatic melanoma cell lines were found to have specific, saturable, high affinity ET-1 binding. Northern analysis and competitive inhibition studies confirmed that melanoma cells express the ETB receptor isoform. Ten nanomolar ET-1 caused an 8.2 to 25.5-fold increase in intracellular free calcium. ET-1 was found to be a weak mitogen for melanoma cells, however, melanoma cell chemokinesis was significantly increased by ET-1. These data suggest that ET-1 may be involved in providing a chemokinetic and growth factor environment that enhances perivascular proliferation and invasiveness of melanoma cells.     

Propofol preserves the viability of isolated rat hepatocyte suspensions under an oxidant stress

OBJECTIVES: This study evaluated the long-term impact of endoluminal low power red laser light (LPRLL) on restenosis in an atherosclerotic rabbit model. BACKGROUND: Despite widespread application of balloon angioplasty for treatment of coronary artery disease, restenosis limits its clinical benefits. Restenosis is a complex process and may be partly attributed to the inability of the vascular endothelium to regenerate and cover the denuded area at the site of arterial injury. We previously demonstrated that LPRLL stimulates endothelial cell proliferation in vitro and contributes to rapid endothelial regeneration after balloon injury in nonatherosclerotic rabbits. METHODS: Rabbit abdominal aortas (n = 12) were treated in separate zones with balloon dilation and balloon dilation plus laser illumination. Endoluminal laser therapy was performed using a laser-balloon catheter delivering a single dose of 10 mW for 3 min from a helium-neon laser (632 nm). Angiography was performed before and after treatment and was repeated 8 weeks before harvesting the aortas. RESULTS: Quantitative angiographic analysis demonstrated no differences in the minimal lumen diameter (MLD) between the two zones before treatment; an increase in the MLD in both zones after balloon angioplasty and a significant versus slight reduction of the MLD in the balloon treatment versus balloon plus laser zones at 8 weeks. Histologic examination showed a very high level of myointimal hyperplasia in the balloon treatment zones but a minimal level in the LPRLL-treated zones. Morphometric analysis revealed a statistically significant difference in the lumen area, intimal area and intima/media ratio between the balloon versus balloon plus laser treatment sites. CONCLUSIONS: Our experimental data indicate that endoluminal irradiation with LPRLL prevents restenosis after balloon angioplasty in an atherosclerotic rabbit model.     

Effect of polynucleotides on the inhibition of neutrophil elastase by mucus proteinase inhibitor and alpha 1-proteinase inhibitor

DNA released from neutrophils at sites of inflammation may modulate tissue proteolysis. We used tRNA and synthetic polynucleotides as models of DNA to study the influence of polynucleotides on the inhibition of neutrophil elastase by its endogenous inhibitors alpha1-proteinase inhibitor (alpha1-PI) and mucus proteinase inhibitor (MPI). Affinity chromatography showed that polynucleotides form electrostatic complexes with elastase and MPI but not with alpha1-PI, the highest affinity being for MPI. The tight-binding partial inhibition of elastase by polynucleotides was used to calculate the Kd of the elastase-polynucleotide complexes which ranged from 4 microM to 21 nM. One mole of tRNA was able to bind 9 mol of elastase. Polydeoxycytosine and tRNA significantly impaired the reversible inhibition of elastase by MPI: they moderately increased the rate of enzyme-inhibitor association, strongly enhanced the rate of complex dissociation, and lowered the enzyme-inhibitor affinity by factors of 34 and 134, respectively. The two polynucleotides also decreased the rate of the irreversible inhibition of elastase by alpha1-PI by factors of 30 and 3, respectively. Polynucleotides also changed the mechanism of inhibition of elastase by the two inhibitors from a one-step inhibition reaction to a two-step binding mechanism. Our data may help explain why proteolysis may occur at sites of inflammation despite the presence of active proteinase inhibitors.     

Splenocaval shunting for alleviation of portal hypertension in a dog: a case report

To demonstrate the usefulness of in vivo 31P nuclear magnetic resonance spectroscopy (MRS) in the diagnosis of ischemic heart disease, major findings from three clinical cardiac MRS investigations performed at our institute are summarized. The first study investigated whether 31P MRS with handgrip exercise testing could detect myocardial ischemia demonstrated by exercise 201Tl scintigraphy. Contrary to findings in normal subjects or patients with fixed thallium defects, the ratio of phosphocreatine (PCr) to ATP decreased significantly during exercise in patients with reversible thallium defects. In the second study, PCr and ATP content was measured by 31P MRS and compared in human myocardium with reversible ischemia or scar diagnosed by exercise thallium scintigraphy. Although the PCr content decreased in patients with either reversible or fixed thallium defects, the ATP content decreased only in the latter group. In the third study, postischemic myocardium with chronic mechanical dysfunction that exhibits recovery after revascularization in left ventriculography was metabolically characterized using quantitative cardiac 31P MRS. Postischemic myocardium with reversible mechanical dysfunction demonstrated reduced PCr but normal ATP content. These results suggest that 31P MRS is a clinically important method both for the detection of myocardial ischemia and in the evaluation of myocardial viability.     

Neurocalcin-immunoreactive cells in the rat hippocampus are GABAergic interneurons

Neurocalcin (NC) is a recently described calcium-binding protein isolated and characterized from bovine brain. NC belongs to the neural calcium-sensor proteins defined by the photoreceptor cell-specific protein recoverin that have been proposed to be involved in the regulation of calcium-dependent phosphorylation in signal transduction pathways. We analyzed the distribution and morphology of the NC-immunoreactive (IR) neurons in the rat dorsal hippocampus and the coexistence of NC with GABA and different neurochemical markers which label perisomatic inhibitory cells [parvalbumin (PV) and cholecystokinin (CCK)], mid-proximal dendritic inhibitory cells [calbindin D28k (CB)], distal dendritic inhibitory cells [somatostatin (SOM) and neuropeptide Y (NPY)], and interneurons specialized to innervate other interneurons [calretinin (CR) and vasoactive intestinal polypeptide (VIP)]. NC-IR cells were present in all layers of the dentate gyrus and hippocampal fields. In the dentate gyrus, NC-IR cells were concentrated in the granule cell layer, especially in the hilar border, whereas in the CA fields they were most frequently found in the stratum radiatum. NC-IR cells were morphologically heterogeneous and exhibited distinctive features of non-principal cells. In the dentate gyrus, pyramidal-like, multipolar and fusiform (horizontal and vertical) cells were found. In the CA3 region most NC-IR cells were multipolar, but vertical and horizontal fusiform cells also appeared. In the CA1 region, where NC-IR cells showed most frequently vertically arranged dendrites, multipolar, bitufted and fusiform (vertical and horizontal) cells could be distinguished. All the NC-IR cells were found to be GABA-IR in all hippocampal layers and regions, and they represented about 19% of the GABA-positive cells. NC/CB, NC/CR and NC/VIP double-labeled cells were found in all hippocampal regions, and represented 29%, 24% and 18% of the NC-IR cells, respectively. NC and CCK did not coexist in the dentate gyrus; however, 9% of the NC-IR cells in the CA fields also contained CCK. No coexistence of NC with PV, SOM or NPY was found in any hippocampal region. We conclude that NC is exclusively expressed by interneurons in the rat hippocampus. NC-IR cells are a morphologically and neurochemically heterogeneous subset of GABAergic non-principal cells, which, on the basis of the known termination pattern of the colocalizing markers, are also functionally heterogeneous and are mainly involved in feed-forward dendritic inhibition in the commissural-associational and Schaffer collateral termination zones (CB containing cells), in innervation of other interneurons (CR- and VIP-containing cells), and in perisomatic inhibition (CCK-containing cells). NC is never present in perisomatic inhibitory PV-containing cells, or in feed-back distal dendritic inhibitory SOM/NPY-containing cells.     

Chronic active hepatitis induced by Helicobacter hepaticus in the A/JCr mouse is associated with a Th1 cell-mediated immune response

Helicobacter hepaticus infection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice to H. hepaticus infection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells to H. hepaticus antigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-gamma) than interleukin-4 (IL-4) or IL-5 when cultured with H. hepaticus outer membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-gamma in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected with Helicobacter pylori and Helicobacter felis, respectively. Mice infected with H. hepaticus developed progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response to H. hepaticus infection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response to Helicobacter.     

Okadaic acid induces selective arrest of protein transport in the rough endoplasmic reticulum and prevents export into COPII-coated structures

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15 degrees C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.