Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.     

Classification of cervical smears with discordance between the cytologic and/or histologic ratings

The problems of diagnostic variability between certified cytotechnologists was studied. Three cytology laboratories submitted a total of 28 cervical smears that had a discordance between the cytologic and/or histologic ratings. Eight independent cytotechnologists provided blind readings on each slide, expressed as "absence of cervical intraepithelial neoplasia (CIN)" to "CIN III." The median rating was absence of CIN or CIN I for 8 slides, CIN II for 5 and CIN III for 15. With a kappa value greater than 0 reflecting agreement beyond chance expectation and a value of 0.40 indicating fair agreement, the kappa value for 8 X 28 ratings was 0.36 (P = .0001), with a 90% confidence interval (CI) between 0.34 and 0.37. The kappa value was 0.14 (P = .10), with a 90% CI between 0.10 and 0.18, on a subsample of nine smears with two or more positive cytology diagnoses but a negative histology. Sixteen of the 28 slides represented cases of histologically proven cancer. Treating cytologic diagnoses of CIN II and CIN III as positive, the sensitivity of the cytologist with reference to histology varied between 71% and 86% while the specificity ranged from 18% to 62%. The positive predictive value was 1/2.5 to 1/1 and the negative predictive value was 1/6 to 1/1. The predictive power (true positives/false positives) ranged from 1.0 to 2.2. The cytodiagnosis of these cervical smears from cases of discordance thus exhibited limited reliability. Standardization of the relevant cytologic knowledge and its routine application is needed to improve the level of performance.     

Correlations between cyclic AMP binding and chemoreception in Paramecium

Paramecium tetraurelia is attracted to cyclic AMP, which probably, as other attractants, signifies the presence of food. Attraction to cyclic AMP was specific, saturable, and, therefore, likely to be receptor-mediated. In these studies, we measured the binding of cyclic [3H]AMP to whole cells and found it to be saturable, reversible, and displaying specificity similar to that of attraction. An HPLC method of separating nucleotides was devised and used to determine that external cyclic AMP was degraded in the absence of IBMX, a phosphodiesterase inhibitor, and that cyclic AMP was taken into the cells in small amounts. Since binding and attraction were subsequently measured in the presence of IMBX, it was cyclic AMP and not a degradation product that served as the attractant stimulus for Paramecium.     

Differentiation between Xanthomonas campestris pv. graminis (ISPP List 1980), pv. phleipratensis (ISPP List 1980) emend., pv. poae Egli and Schmidt 1982 and pv. arrhenatheri Egli and Schmidt 1982, by Numerical Analysis of Phenotypic Features and Protein Gel Electrophoregrams

A numerical analysis of 257 phenotypic features of 45 bacterial isolates from grasses, revealed three phenons corresponding to (i) X. campestris pv. graminis (ISPP List 1980), (ii) X. campestris pv. phleipratensis (ISPP List 1980) and (iii) X. campestris pv. poae Egli and Schmidt 1982 and X. campestris pv. arrhenatheri Egli and Schmidt 1982. In each phenon, the strains clustered together regardless of the geographical origin of the isolates orthe year of isolation. Polyacrylamide gel electrophoresis of soluble proteins and host range studies, revealed four groups corresponding to the pathovars mentioned above. The four pathovars constitute definite biological entities that can be differentiated by phenotypic, gel electrophoretic and host range features.     

Detection by ELISA of Two Tobamoviruses in Orchids Using Monoclonal Antibodies

Five monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus-coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS-ELISA. None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal-monoclonal antibody DAS-ELISA system is advocated for detecting both tobamoviruses in orchids.     

Diurnal variation in 5′-nucleotidase activity in rat liver

Summary The diurnal variation of 5-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora.

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.     

Inhibition of human neutrophil collagenase by gold(I) salts used in chrysotherapy

Six gold(I) salts, some of which are used as drugs in chrysotherapy, are shown to be inhibitors of two forms of human neutrophil collagenase. The IC50 values vary over six orders of magnitude, the lowest being 3.5 nM for Myocrisin. Thus, inhibition is greatly affected by the identity of the ligands to the gold(I) atom. The inhibition of collagenase by these gold(I) salts may be a partial basis for their antiarthritic action.     

Prevalence of polymorphic 21-hydroxylase gene (CA21HB) mutations in salt-losing congenital adrenal hyperplasia

Using genomic restriction analysis of 14 unrelated patients with salt-losing congenital adrenal hyperplasia, we identified three different CA21HB mutation patterns: no detectable restriction fragment abnormalities (16/28 haplotypes), deletion of the active CA21HB gene (9/28), and apparent conversion of the active CA21HB gene to the pseudogene CA21HA (3/28). CA21HB gene deletion was associated with HLA-Bw47 in 6 haplotypes and with absent C4B expression in 7. A variety of HLA and C4 types was associated with the other mutations. Apparent conversion of CA21HB to CA21HA was identified by the disparity between the intensity ratios for the major TaqI and BglII hybridization fragments.