Contribution of the cytoskeleton and the phospholipase C signaling pathway to fluid stream-induced membrane currents

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I(i,st)) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I(i,st) were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I(i,st), suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP(3))-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I(i,st) in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I(i,st). The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP(3) concentration, in turn releasing calcium from InsP(3)-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.     

Novel O-glycosyl amino acid mimetics as building blocks for O-glycopeptides act as inhibitors of galactosidases

As potential lead structures for a new class of glycosidase inhibitors the novel O-glycosyl amino acid mimetics 3'-O-[2,6-anhydro-D-glycero-L-gluco-heptitol-1-yl]-L-serine 3 and-L-threonine 4 were synthesized, employing regio- and stereoselective aziridine ring opening methodology. They proved to be stable in the presence of glycosidases and showed competitive inhibition of alpha-galactosidase from Aspergillus niger.     

Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites

In extracts of senescent leaves of the tobacco plant Nicotiana rustica, two colorless compounds with UV/VIS characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively identified as Nr-NCCs. These two polar NCCs were found in similar amounts in the fresh extracts, and their constitutions could be determined by spectroscopic analysis. The data showed both of the two Nr-NCCs to have the same tetrapyrrolic core structure, as reported previously for all other NCCs from senescent higher plants. In the less polar catabolite, named Nr-NCC-2, this core structure was conjugated with a glucopyranose unit, as similarly discovered earlier in Bn-NCC-2, an NCC from oilseed rape (Brassica napus). The more polar NCC from tobacco leaves, Nr-NCC-1, carried an additional malonyl substituent at the 6'-OH group of the glucopyranosyl moiety. Partial (enzyme-catalyzed) hydrolysis of Nr-NCC-1 gave Nr-NCC-2, while enzyme-catalyzed malonylation of Nr-NCC-2 gave Nr-NCC-1, establishing the identity of their basic tetrapyrrole structure. In earlier work (on the polar NCCs from oilseed rape), only separate glucopyranosyl and malonyl functionalities were detected. Nr-NCC-1, thus, represents a further variant of the structures of NCCs from senescent higher plants and exhibits an unprecedented peripheral refunctionalization in chlorophyll catabolites.     

Physiological effects of lactulose and inulin in the caecum of rats

A model experiment was performed on rats to evaluate the effect of partial or total substitution of saccharose (S) and cellulose (C) by preparations of lactulose and inulin on the development and metabolism of the caecum. In the experimental diets given to rats for 4 weeks, the examined preparations were administered either with an equivalent amount of cellulose (each at 4% of the diet) or as sole source of dietary fibre at 8% of the diet. Compared to the saccharose group cellulose had no effect, and low doses of lactulose and inulin in the diet increased to a medium extent the weight of the caecum wall and caecal digesta. The addition of lactulose and inulin at 8% increased significantly the content of caecal digesta (4.62 and 4.11 g/100g BW, respectively) and the weight of the caecal wall (1.10 and 0.86 g/100g BW, respectively), compared to the groups with saccharose and cellulose (0.73, 0.90 and 0.24, 0.28 g/100g BW, respectively). Cellulose and cellulose partially-substituted with lactulose and inulin caused an increase in the dry matter content of caecal digesta (26.5-27.5%), compared to other groups (21.8-22.8%). The administration of lactulose and inulin preparations was accompanied by a significant drop in pH (5.47-5.81), compared to the groups with cellulose or saccharose (6.83-6.91), and a decrease in the ammonia concentration in the caecal digesta, compared to the cellulose control (0.27-0.40 and 0.62 mg/g, respectively). The group with 8% lactulose was characterized by the highest activities of microbiological alpha- and beta-galactosidase and beta-glucosidase in the caecal digesta. Cellulose and both preparations significantly decreased the activity of beta-glucuronidase, compared to the saccharose group (0.39-0.89 and 1.52 U/g, respectively). The highest concentration of VFA in the caecal digesta was observed in the saccharose group (89.2 micromol/g), and the lowest concentration in the group where cellulose was totally substituted by lactulose and inulin (55.1 and 57.5 micromol/g, respectively). The total production of VFA in the caecum was fourfold higher with 8 % lactulose and inulin (254.7 and 236.4 micromol/100g BW, respectively) than in both controls groups (65.1 and 67.8 micromol/100g BW, respectively). The high dose of inulin and lactulose increased the share of propionic acid in the VFA profile (C2:C3:C4) compared to both control groups. When 4% inulin was added to the diet a significant increase of butyrate concentration in the caecum was observed.     

Angiotensin II feedback is a regulator of renocortical renin,COX-2, and nNOS expression

Salt restriction leads to parallel increases of renin, cyclooxygenase-2 (COX-2), and neuronal nitric oxide synthase (nNOS) gene expression in the juxtaglomerular apparatus of rat kidneys. Because the upregulation of these genes is strongly enhanced if salt restriction is combined with inhibition of the renin-angiotensin-aldosterone system, our study aimed to find out whether the juxtaglomerular expressions of renin, COX-2, and nNOS are subject to a common direct negative feedback control by ANG II. For this purpose, male Sprague-Dawley rats were fed a low-salt diet (0.02% wt/wt) with or without additional treatment with the ANG I-converting enzyme (ACE) inhibitor ramipril (10 mg x kg body wt(-1) x day(-1)) for 1 wk, and renocortical renin, COX-2, and nNOS mRNAs were assayed. To narrow down possible indirect effects of the ACE inhibitor that may result from insufficient aldosterone production, the animals received mineralocorticoid substitution with fludrocortisone (6 mg. kg body wt(-1) x day(-1)). Thus mineralocorticoid substitution prevented the fall of systolic blood pressure and of glomerular filtration induced by ramipril in rats on low-salt diet. Although fludrocortisone had no effect on basal renin, COX-2, and nNOS mRNA, it clearly attenuated the threefold increases of both renin and COX-2 mRNA in response to low-salt diet. In rats on low-salt diet, ramipril further increased renin mRNA ninefold, COX-2 mRNA fourfold, and nNOS 2.5-fold in the absence of fludrocortisone. In the presence of fludrocortisone, ramipril increased renin mRNA 10-fold, COX-2 mRNA 2.5-fold, and nNOS mRNA 2.5-fold. These data indicate that mineralocorticoid substitution lowers the overall expression of juxtaglomerular renin and COX-2 during low-salt intake and attenuates a further rise of COX-2 expression by ACE inhibition, but it does not change the stimulatory effect of ACE inhibition on renin and nNOS expression. We conclude that the expression of renin, COX-2, and nNOS in the juxtaglomerular apparatus during low-salt diet is markedly limited by a direct feedback inhibition through ANG II.     

Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.     

EcoRII: a restriction enzyme evolving recombination functions?

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5'CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two-domain structure that enables this particular mode of protein-DNA interaction. The C-terminal domain is a new restriction endonuclease, EcoRII-C. In contrast to the wild-type enzyme, EcoRII-C cleaves DNA specifically at single 5'CCWGG sites. Moreover, substrates containing two or more cooperative 5'CCWGG sites are cleaved much more efficiently by EcoRII-C than by EcoRII. The N-terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5'CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA-binding domain to enable the interaction with two 5'CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons.