Ionic mechanisms of action of neurotensin in acutely dissociated neurons from the diagonal band of Broca of the rat

Whole cell recordings were performed on acutely dissociated neurons from the horizontal limb of the diagonal band of Broca (hDBB) from rats to elucidate the ionic mechanisms of action of neurotensin. Neurotensin caused a decrease in whole cell voltage-activated outward currents and failed to elicit a response when Ca2+ influx was blocked by changing the external solution to the one containing 0 mM Ca2+ and 50 microM Cd2+, suggesting the involvement of Ca2+-dependent conductances. Charybdotoxin, a specific blocker of voltage-sensitive calcium-activated K+ channels (IC), caused a decrease in outward currents comparable with that caused by blocking calcium influx and occluded the neurotensin-induced decrease in outward currents. Similarly, 50 microM tetraethylammonium ions also blocked the neurotensin response. Also neurotensin reduced whole cell barium currents (IBa) and calcium currents (ICa). Amiloride and omega-conotoxin GVIA, but not nimodipine, were able to eliminate the neurotensin-induced decrease in IBa. Thus T- and N- but not L-type calcium channels are subject to modulation by neurotensin, and this may account for its effects on IC. The predicted changes in action potential as a result of the blockade of currents through calcium channels culminating into changes in IC were confirmed in the bridge current-clamp recordings. Specifically, neurotensin application led to depolarization of the resting membrane potential, broadening of spike and a decrease in afterhyperpolarization and accommodation. These alterations in action potential characteristics that resulted in increased firing rate and excitability of the hDBB neurons also were produced by application of charybdotoxin. Neurotensin effects on these properties were occluded by 2 - [(1 - 7 - chloro - 4 - quinolinyl) - 5 - (2, 6 - di - methoxyphenyl) pyrazol-3-yl) carbonylamino] tricyclo (3.3.1.1.)decan-2-carboxylic acid, a nonpeptide high-affinity neurotensin receptor antagonist. Neurotensin blockade of IC, possibly through ICa, is a potential physiological mechanism whereby this peptide may evoke alterations in the cortical arousal, sleep-wake cycle, and theta rhythm.     

Supplementary anti-hepatitis C virus (HCV) testing with 2nd and 3rd generation recombinant immunoblot assay and matrix applied to enzyme immunoassay positive sera and comparison with HCV-RNA detection

Following the same general principles of its two predecessors in 1973 and 1986, the 1996 National Roadside Survey of weekend, nighttime drivers in the 48 contiguous states interviewed and breath tested over 6000 noncommercial four-wheel vehicle operators between September 6 and November 9, 1996. Results indicated that the total number of drinking drivers fell by about one-third between 1986 and 1996; however, there was no significant change in the number of drivers at blood alcohol concentrations (BACs) at or above 0.05. Compared to 1973, the proportion of women drivers on the roads during weekend nights has increased significantly. Moreover, relative to males, the proportion of female drivers who have been drinking has increased over the last decade. The number of drivers under the age of 21 with a BAC at or above 0.10 decreased significantly from 1986 to 1996.     

Diffusion coefficients in the lateral intercellular spaces of Madin-Darby canine kidney cell epithelium determined with caged compounds

The diffusion coefficients of two caged fluorescent dyes were measured in free solution and in the lateral intercellular spaces (LIS) of cultured Madin-Darby canine kidney (MDCK) cells after photoactivation by illumination with a continuous or pulsed UV laser. Both quantitative video imaging and a new photometric method were utilized to determine the rates of diffusion of the caged fluorescent dyes: 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)pyrene-1,3,6-trisulfonic acid (DMNB-HPTS) and (4,5-dimethoxy-2-nitrobenzyl) fluorescein dextran (10,000 MW) (DMNB-caged fluorescein dextran). The diffusion coefficients at 37 degrees C in free solution were 3.3 x 10(-6) cm2/s (HPTS) and 0.98 x 10(-6) cm2/s (10,000 MW dextran). Diffusion of HPTS within nominally linear stretches of the LIS of MDCK cells grown on glass coverslips was indistinguishable from that in free solution, whereas dextran showed a 1.6 +/- 0.5-fold reduction in diffusivity. Measurements of HPTS diffusion within the LIS of multicellular regions also exhibited a diffusivity comparable to the free solution value. The restriction to diffusion of the dextran within the LIS may be due to molecular hindrance.     

Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor

Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.     

The action of blocking agents applied to the inner face of Ca(2+)-activated K+ channels from human erythrocytes

The 'capacity' of the vagina and the compliance of the vaginal wall was measured in bilaterally ovariectomized ewes (n = 7) before and after treatment with exogenous oestradiol and progesterone; in nulliparous ewes (n = 7) at oestrus (Day 0) and dioestrus (Day 10) and during pregnancy, and in another group of pregnant ewes (n = 15) treated with exogenous oestradiol and progesterone. Measurements were remarkably consistent within individual animals but there were considerable differences between individual animals. The 'vaginal capacity' and the compliance of the vaginal wall were greater at oestrus than during dioestrus. In the same seven ewes, which were studied during their first and second pregnancies, the 'capacity' of the vagina increased whereas the compliance of the vaginal wall declined; from 90 days to term both parameters remained fairly constant. For the first 2 months of gestation the vaginal capacity was greater in year 2 than year 1 but this was reversed during the last 3 months. The compliance of the vaginal wall was significantly greater (P < 0.0001) in year 2 than year 1 at all stages of pregnancy. In ovariectomized ewes, progesterone only significantly increased the vaginal capacity at the highest dose rate (viz. 100 mg); the compliance of the wall was reduced at the 25 and 50 mg dose rates. Oestradiol produced an inconsistent dose response effect; whilst 5 mg and 20 mg had no effect upon the vaginal capacity, the 10 mg dose rate significantly reduced it. Similarly, the highest and lowest dose rates reduced the compliance of the vaginal wall but the 10 mg dose rate increased it. At 90 and 120 days of gestation, both 5 mg oestradiol and 100 mg progesterone increased the vaginal capacity but reduced the compliance.     

A comparison of methods of phenotypic and genotypic fingerprinting of Exophiala dermatitidis isolated from sputum samples of patients with cystic fibrosis

A 77-year-old man was admitted because of massive pericardial effusion and cardiac tumor. Cytological examination of the effusion and histological examination of a subcutaneous tumor in the chest wall revealed diffuse large B cell lymphoma. The immunophenotype of tumor cells was CD5+ CD20+ CD22+ CD38+ HLA-DR+ CD19-. Chromosome analysis revealed complex abnormal karyotypes containing t(8;14) (q24;q32). C-myc gene rearrangement was shown by Southern blotting. Chemotherapy with pirarubicin, cyclophosphamide, vincristin, and prednisolone (THP-COP) was not effective for his lymphoma. He suffered from cardiac tamponade and died at 5 months after diagnosis. Autopsy revealed a large cardiac tumor, extensive epicardial infiltration, tiny tumors in the lung and pancreas, but no lymphadenopathy, the combination of which suggested a primary cardiac lymphoma. Immunohistochemistry for p53 protein showed nuclear staining of more than 50% of the lymphoma cells. In situ hybridization for EBER-1 was negative. Rearrangement of c-myc gene and overexpression of p53 protein are usually observed in Burkitt's lymphoma and some cases of high grade lymphomas including AIDS-associated non-Hodgkin lymphomas. In this case the association of these molecular findings and resistance to chemotherapy is suggested.