The effect of 2-amino-4-phosphonobutyrate (APB) on acetylcholine release from the rabbit retina: evidence for on-channel input to cholinergic amacrine cells

The light-evoked release of acetylcholine (ACh) from the rabbit retina was taken as a measure of cholinergic amacrine cell activity. The glutamate analogue DL-(+/-)-2-amino-4-phosphonobutyric acid (APB) prevented the light-evoked release of ACh and also selectively abolished the ON-responses of ganglion cells and the ERG b-wave. It is concluded that the input to cholinergic amacrine cells involves mainly the depolarizing bipolar cells, which subserve ON-channels. L-(+)-stereoisomer of APB was 15 times more potent than the D-(-)-isomer in suppressing ACh release and the b-wave, suggesting that the mechanism of action of APB does not involve antagonism of excitatory amino acids.     

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.     

Gluthatione-S-transferase P1 polymorphism I105V in familial and sporadic prostate cancer

Several reports suggest that the glutathione-S-transferase (GST) family of enzymes is involved in a variety of cancers, due to their carcinogen-detoxification properties. A polymorphism in codon 105 of the pi variant (GSTP1 I105V), which affects the enzymatic activity of the enzyme, has been linked to the incidence of cancers from different organs. However, the published data in prostate cancer (PCa) is controversial. Some studies report an association with the GSTP1 I105V polymorphism and sporadic PCa, whereas other studies report no association. Recently, one study showed a positive correlation between the GSTP1 I105V polymorphism and familial PCa in a Japanese population. In the present study, we assessed the correlation of the GSTP1 I105V polymorphism with familial and sporadic PCa in an American population. We analyzed DNA samples from 438 patients with familial PCa, 499 patients with sporadic PCa, and 510 controls. We found no significant association between the GSTP1 I105V polymorphism and familial or sporadic PCa when compared to the control group [odds ratio (OR) =1.0 (0.74-1.37); P=0.58]. Moreover, no association was found after stratification for age of diagnosis, Gleason grade, or lymph node involvement [OR =0.84 (0.65-1.09), P=0.37]. These data indicate that there is no associated risk for sporadic or familial PCa in American families containing the GSTP1 I105V polymorphism.     

Vesicular targeting and the control of ion secretion in epithelial cells: implications for cystic fibrosis.

Non-polarized HT-29 colonic epithelial cells fail to respond to cyclic AMP-generating agonists with increases in plasma membrane anion conduction. Radio-isotopic efflux and patch-clamp experiments revealed that both undifferentiated and differentiated HT-29 colonocytes possess volume- and Ca(2+)-activated Cl- channels. However, only within the apical plasma membranes of the latter were cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels found. CFTR was expressed equally well in both non-polarized and polarized colonocytes. Lack of CFTR-dependent anion conduction was shown to be the result of CFTR retention within a peripheral intracellular compartment. We demonstrate that upon polarization, CFTR moves to the apical plasma membrane via a Brefeldin A (BFA)-sensitive intracellular trafficking pathway.     

Routine antenatal screening for hepatitis B using pooled sera: validation and review of 10 years experience.

AIMS: To validate the sensitivity of universal antenatal screening for hepatitis B surface antigen (HBsAg) by testing pools of 10 sera, and to review 10 years' experience using this method. METHODS: 66,945 antenatal patients were tested between 1986 and 1996 using the pooled method. All sera from 1996 (n = 6050) were retrieved and retrospectively tested individually. An in vitro determination of the effect of pooling on sensitivity was performed by checkerboard neutralisation assay. RESULTS: 26 HBsAg positive women were detected by universal screening over 10 years; 12 had non-European surnames and five had known risk factors for hepatitis B infection. High titre anti-HBs sera in the pool reduced the sensitivity of the HBsAg assay, though the effect was only significant at low levels of HBsAg carriage. CONCLUSIONS: The prevalence of hepatitis B is extremely low in the antenatal population served by Plymouth PHL. Pooling is unlikely to reduce sensitivity enough to lead to significant preventable vertical transmission, and is a cost-effective and valid strategy in areas of low seroprevalence.     

The off-transient pulmonary oxygen uptake (VO(2)) kinetics following attainment of a particular VO(2) during heavy-intensity exercise in humans

The oxygen uptake response to moderate-intensity exercise (i.e. < anaerobic threshold (an)) has been characterised with a gain (i.e. response amplitude per increment of work rate) and time constant that do not vary appreciably at different work rates or between the on- and off-transients. Above an, the response becomes more complex with an early component that typically projects to a value that has a gain similar to that of the < an response, but which is supplemented by the addition of a delayed slow kinetic component. We therefore established a constant target VO2 (VO21) for each subject such that with different imposed work rates the contribution to VO21 from the slow phase varied over a wide range. Work rates were chosen so that VO21 was attained at 2-24 min. Five subjects (aged 21-58 years) cycled at four to five different work rates. VO2 was measured breath-by-breath, at VO21 the work rate was abruptly reduced and the subject recovered by cycling unloaded for 15 min. Unlike the on-transient, for which the slow component shows a long delay, the off-transient was best fitted as two simultaneous exponential components. The slower off-transient component had a small amplitude and long time constant, but did not differ significantly among the various tests. The off-transient kinetics for VO2 therefore was independent of the magnitude of the contribution to the slow phase from the on-transient kinetics.     

Ethanol-induced conditioned place preference is expressed through a ventral tegmental area dependent mechanism

The authors examined the role of the ventral tegmental area (VTA) and nucleus accumbens (NAc) in the expression of ethanol-induced conditioned place preference (CPP). After cannulas were implanted, male DBA/2J mice underwent an unbiased Pavlovian-conditioning procedure for ethanol-induced CPP. Before preference testing, the mice were injected intra-VTA (Experiments 1 and 3) or intra-NAc (Experiment 2) with the nonselective opioid antagonist methylnaloxonium (0-ng, 375-ng, or 750-ng total infusion; Experiments 1 and 2) or the gamma aminobutyric acid (GABA(B)) agonist baclofen (0-ng, 25-ng, or 50-ng total infusion; Experiment 3). Intra-VTA methylnaloxonium or baclofen decreased ethanol-induced CPP, whereas intra-NAc methylnaloxonium had no effect. These findings indicate that the conditioned rewarding effect of ethanol is expressed through a VTA-dependent mechanism that involves both opioid and GABA(B) receptors.     

Ability of bacteria associated with chronic inflammatory disease to stimulate E-selectin expression and promote neutrophil adhesion.

Porphyromonas gingivalis, Pseudomonas aeruginosa, and Helicobacter pylori have been shown to be associated with adult periodontal disease, chronic lung infections, and peptic ulcers, respectively. The ability of these bacteria to stimulate E-selectin expression and promote neutrophil adhesion, two components necessary for the recruitment of leukocytes in response to infection, was investigated. Little or no stimulation of E-selectin expression was observed with either P. gingivalis or H. pylori when whole cells, lipopolysaccharide (LPS), or cell wall preparations added to human umbilical cord vein endothelial cells were examined. P. aeruginosa was able to induce E-selectin to near-maximal levels; however, it required approximately 100 to 1,000 times more whole cells or LPS than that required by E. coli. Neutrophil-binding assays revealed that LPS and cell wall preparations obtained from these bacteria did not promote endothelial cell adhesiveness by E-selectin-independent mechanisms. In addition, P. gingivalis LPS blocked E-selectin expression by LPS obtained from other bacteria. We propose that lack of E-selectin stimulation and the inability to promote endothelial cell adhesiveness are two additional indications of low biologically reactive LPS. We suggest that this property of LPS may contribute to host tissue colonization. In addition, the ability of P. gingivalis to inhibit E-selectin expression may represent a new virulence factor for this organism.     

Immunoblot analysis of anti-Ureaplasma urealyticum antibody in pregnant women and newborn infants.

The purpose of the present study was to determine the frequency in which antibody reactive to Ureaplasma urealyticum could be detected in a population of pregnant women and newborn infants. Serum samples from a prospective cohort of 80 healthy, U. urealyticum culture-positive and culture-negative pregnant women and a retrospective cohort of 522 infants born at between 25 and 42 weeks of gestation were studied by immunoblot analysis. Cultures of specimens from the lower genital tract were positive for U. urealyticum for 83% of the pregnant women, and serum immunoglobulin G (IgG) antibody which reacted to U. urealyticum was detectable in 93% of the pregnant women. Samples from five women (8%) had increases in the number of anti-U. urealyticum IgG bands over the course of the pregnancy. Samples from four of these five women had corresponding increases in the number of antibody bands present in IgA immunoblots. Six of the 522 samples from newborns or cord blood (1.1%) were positive for anti-U. urealyticum IgA; 5 of these 6 samples were also positive for IgM. The six anti-U. urealyticum IgA-positive infants were distributed as follows; 3 of 67 (4.5%) infants were delivered at 25 to 30 weeks of gestation, 3 of 176 (1.7%) infants were delivered at 31 to 34 weeks of gestation, and 0 of 279 infants were delivered at > or = 35 weeks of gestation. An antibody response to U. urealyticum can be detected in pregnant women and preterm infants and may serve as a marker of infection.     

Microcarrier culture of hepatocytes in whole plasma for use in liver support bioreactors.

Contact activation of plasma clotting may limit the use of some microcarrier types for hepatocyte attachment in a liver assist device. Activation by seven microcarrier types was studied in plasma containing 2-500 units heparin/mL. Clotting was activated by dextran (Cytodex 1 and 2) and collagen-coated (Cytodex 3) microcarriers at 2-25 units/mL (Cytodex 1) and 2-100 units/mL (Cytodex 2 and 3). There was no activation by polystyrene, gelatin, glass or fibronectin-coated polystyrene microcarriers. Compared with culture medium, incubation of HepG2 cells in plasma did not affect cell viability but increased cell number (56.4 versus 65.1 x 10(4) cells; P less than 0.05) and incorporation of [3H]-amino acids into protein (204913 versus 279624 dpm; P less than 0.05). Polystyrene-attached cells demonstrated time-linear protein synthesis, glucose and 7-ethoxycoumarin metabolism. We conclude that polystyrene-attached hepatocytes maintain viability and metabolic activity in plasma and are of potential use in a liver support bioreactor.