Independent trafficking of KATP channel subunits to the plasma membrane

KATP channels are unique in requiring two distinct subunits (Kir6.2, a potassium channel subunit) and SUR1 (an ABC protein) for generation of functional channels. To examine the cellular trafficking of KATP channel subunits, green fluorescent protein (GFP) was tagged to the cytoplasmic N or C terminus of SUR1 and Kir6. 2 subunits and to the C terminus of a dimeric fusion between SUR1 and Kir6.2 (SUR1-Kir6.2). All tagged constructs generated functional channels with essentially normal properties when coexpressed with the relevant other subunit. GFP-tagged Kir6.2 (Kir6.2-GFP) showed perinuclear and plasma membrane fluorescence patterns when expressed alone or with SUR1, and a very similar pattern was observed when channel-forming SUR1-Kir6.2-GFP was expressed on its own. In contrast, whereas SUR1 (SUR1-GFP) also showed a perinuclear and plasma membrane fluorescence pattern when expressed alone, an apparently cytoplasmic fluorescence was observed when coexpressed with Kir6.2 subunits. The results indicate that Kir6.2 subunits traffic to the plasma membrane in the presence or absence of SUR1, in contradiction to the hypothesis that homomeric Kir6.2 channels are not observed because SUR1 is required as a chaperone to guide Kir6.2 subunits through the secretory pathway.     

Estimation of the mutagenic potential of the trematode Opisthorchis felineus in experimentally infected guinea pigs

We studied the mutagenic potential of the trematode Opisthorchis felineus in experimentally infected guinea pigs by chromosome analysis. The investigation was conducted on 30 treated (experimentally infected) and 20 normal (uninfected) guinea pigs (Cavia porcellus). A sample of living metacercariae of O. felineus was obtained from the muscle of naturally infected crucian carp captured in the Tom River, and 1 ml suspension containing approximately 50 metacercariae in 0.9% NaCl solution was individually given p.o. to guinea pigs that were used as treated specimens, whereas normal guinea pigs from the same stock were given 1 ml 0.9% NaCl solution per individual to serve as controls. The treated and control animals were killed by CO2 asphyxiation and decapitation at 7, 15, 30, 45, and 120 days after contamination. The smears of bone marrow cells for chromosome analysis were prepared by a standard method. Statistical analysis of the data was done using Student's t-test. We found that experimental infection of guinea pigs with O. felineus induced significantly high frequencies of hypoploid cells and, at the 120th day p.i., led to a considerable incidence of chromatid breaks and polyploidy in bone marrow cells.     

Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene

Leukotrienes constitute a class of potent biological mediators of inflammation and anaphylaxis (for reviews see refs 1 and 2). Their biosynthesis derives from 5-lipoxygenase-catalysed oxygenation of arachidonic acid in granulocytes, macrophages and mast cells. To examine the physiological importance of leukotrienes, we have disrupted the 5-lipoxygenase gene by homologous recombination in embryonic stem cells. 5-Lipoxygenase-deficient (5LX-/-) mice develop normally and are healthy. They show a selective opposition to certain inflammatory insults. Although there is no difference in their reaction to endotoxin shock, the 5LX-/- animals resist the lethal effects of shock induced by platelet-activating factor. Reaction to ear inflammation induced by phorbol ester is normal, whereas inflammation induced by arachidonic acid is markedly reduced. Contrasts were also found in two models of leukocyte chemotaxis in vivo. The phenotype of 5LX-/- mice under injurious insult identifies the role for leukotrienes in the pathophysiology of select inflammatory states.     

Permissive effect of nitric oxide in arachidonic acid induced dilation in isolated rat arterioles

OBJECTIVE: Prostaglandins and nitric oxide play an important role in the regulation of arteriolar tone. L-Arginine analogues inhibit nitric oxide formation, but may also inhibit arachidonic-acid induced dilation. Nitric oxide was found to stimulate cyclooxygenase activity in cultured endothelial cells. Therefore, we hypothesized that the non-specific inhibition of prostaglandin-related dilation by L-arginine analogues is a consequence of the absence of nitric oxide. METHODS: To test this hypothesis, arteriolar segments from rat cremaster muscle were studied in a pressure myograph at 75 mmHg. Segments developed spontaneous tone, the diameter reduced from 179 +/- 3 to 98 +/- 3 microns (n = 41). In this condition, responses to exogenous arachidonic acid (1 microM) were recorded and compared with responses after addition of L-NNA, and addition of either SNAP, nitroprusside or 8-Br-cGMP in the presence of L-NNA. RESULTS: Inhibition of basal nitric oxide production with L-NNA (0.1 mM) reduced arachidonic acid-induced dilation (from 52 +/- 9 to 31 +/- 6 microns). In the presence of L-NNA, responses to arachidonic acid were augmented when exogenous nitric oxide was also present (SNAP, 31 +/- 6 microns vs. 75 +/- 5 microns; nitroprusside, 31 +/- 8 microns vs. 42 +/- 7 microns). Responses were not augmented with the second messenger of nitric oxide-mediated dilation 8-Br-cGMP (37 +/- 9 microns vs. 32 +/- 9 microns). CONCLUSIONS: These results indicate that nitric oxide directly increases arachidonic acid-induced dilation. Thus, the non-specific effect of L-arginine analogues can be explained by a permissive effect of nitric oxide on endothelial arachidonic acid metabolism.     

Determination of hydroperoxides and structures by high-performance liquid chromatography with post-column detection with diphenyl-1-pyrenylphosphine

OBJECTIVE: The aim of this meta-analysis was to review the existent evidence on the effectiveness of tolrestat in the treatment of diabetic peripheral neuropathy. RESEARCH DESIGN AND METHODS: Individual patient data on 738 subjects from the three randomized clinical trials published on this topic were analyzed using changes in motor nerve conduction velocities (NCVs) as endpoints. Nerves investigated included median, ulnar, tibial, and peroneal. RESULTS: The pooled analysis of NCV taken as a continuous measurement showed a significant treatment effect, the magnitude of this benefit being approximately equal to 1 m/s for all the nerves investigated. When looking at the proportion of patients experiencing a loss of NCV of at least 1 or 2 m/s in at least two out of the four nerves investigated, it emerged that treatment reduced by > 40% the risk of such outcomes after adjusting for patients' characteristics. The odds ratios relative to the placebo group were 1.82 (1.30-2.52) and 1.70 (1.15-2.48) for a decrease of 1 and 2 m/s, that is, placebo-treated patients have an 82 and 70% increased risk for a loss of nerve function of 1 and 2 m/s, respectively. No statistically significant difference in treatment effect emerged after stratification according to baseline motor NCV and glycated hemoglobin levels. CONCLUSIONS: After a treatment duration ranging between 24-52 weeks, patients treated with tolrestat had a reduced risk for developing nerve function loss compared with placebo-treated patients. Future long-term trials are needed to evaluate the impact of the treatment on more clinically meaningful endpoints such as the development of foot complications.     

Application of free-flow electrophoresis for isolation and purification of proteins and peptides

Free-flow electrophoresis (FFE) has been applied to the separation and purification of a variety of proteins and polypeptides: bee venom, tumor necrosis factor, interleukin-1beta, interferon-gamma and superoxide dismutase. FFE at constant pH and conductivity of the carrying buffer is shown to be efficient at various separation schemes. In some cases, the method allows us to obtain proteins with a purity of more than 90% at a productivity of 20-30 mg/h. An electrophoretic apparatus with a new, multi-sectional construction of the electrophoretic chamber and a system for cross-displacement of carrying buffer in the chamber is described.     

Efficiency of computed tomography in diagnosis of silicotuberculosis

Seventy-five newborns from multiple pregnancies with very low and extremely low birth weight are studied. 94% of the infants under 1000 g and 66% of those above 1000 g are born by vaginal way. Intrapartal asphyxia develop most often the second twins with birth weight under 1000 g--64.7%. These are the infants with higher morbidity: RDS--56.5%, IVH--100%. The survival rate of the twins of this group is notably lower than that of the infants from singleton pregnancies with equal weight and gestational age: 12.1% under 1000 g and 69% above 1000 g, against 38.3% and 77.9% respectively.