Analysis of pyridine dinucleotides in cultured rat hepatocytes by high-performance liquid chromatography

An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 M perchloric acid or 0.5 N sodium hydroxide containing 50% (v/v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C18 (4.6 X 150 mm) column with 0.1 M potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD+ and NADP+ were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD+ and NADP+ of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400-700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

Australine [(1R,2R,3R,7S,7aR)-3-(hydroxymethyl)-1,2,7-trihydroxypyrrolizid ine] is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis [Molyneux et al. (1988) J. Nat. Prod. (in press)]. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, we tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the alpha-glucosidase amyloglucosidase (50% inhibition at 5.8 microM), but it did not inhibit beta-glucosidase, alpha- or beta-mannosidase, or alpha- or beta-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc3Man7-9(GlcNAc)2-oligosaccharides.     

Excision Repair of Uv Radiation-Induced DNA Damage in Caenorhabditis Elegans

Radioimmunoassays were used to monitor the removal of antibody-binding sites associated with the two major UV radiation-induced DNA photoproducts [cyclobutane dimers and (6-4) photoproducts]. Unlike with cultured human cells, where (6-4) photoproducts are removed more rapidly than cyclobutane dimers, the kinetics of repair were similar for both lesions. Repair capacity in wild type diminished throughout development. The radioimmunoassays were also employed to confirm the absence of photoreactivation in C. elegans. In addition, three radiation-sensitive mutants (rad-1, rad-2, rad-7) displayed normal repair capacities. An excision defect was much more pronounced in larvae than embryos in the fourth mutant tested (rad-3). This correlates with the hypersensitivity pattern of this mutant and suggests that DNA repair may be developmentally regulated in C. elegans. The mechanism of DNA repair in C. elegans as well as the relationship between the repair of specific photoproducts and UV radiation sensitivity during development are discussed.     

Antigen expression from the ribosomal DNA repeat unit of Giardia intestinalis.

The amitochondrial human intestinal parasite Giardia intestinalis is regarded to be the most ancient living example of single-celled eukaryotes and should display primitive features of pre-metazoan gene regulation. Characterization of E. coli clones which express Giardia antigens from plasmid vectors has revealed that an antigen is encoded by the rDNA repeat unit from the strand complementary to that encoding the rRNAs. The open reading frame (ORF) originates in the spacer region between the small (SS) and large (LS) subunit rRNA genes and terminates within the LS rRNA gene. The promoter region of this ORF has characteristics of both RNA polymerase (pol) II and pol III regulatory sequences, suggestive of gene regulation before these different promoter types evolved. The rDNA repeat unit is located on multiple chromosomal sites which are different in each isolate, although the electrophoretic karyotypes appear very stable in Giardia from both human and animal sources.     

SAPID tools: microcomputer programs for analysis of multi-unit nerve recordings

A set of programs is described for the digitization and analysisof electrophysiological recordings in which the nerve impulsesfrom several different cells may be present. Although they weredesigned for analysis of data from insect taste sensilla, theymay be applicable to other multi-unit preparations, and areavailable free from the authors. The programs run on standardMS-DOS compatible microcomputers, using a readily availableanalog-to-digital plug-in board. They are ‘modular’,and break the analysis into several stages, each of which maybe applied to many related files of data in a ‘batch’mode. Program design stresses the involvement of the user indecisions as to the effectiveness and accuracy of the analysisas it proceeds, as well as ease and efficiency of use. The programsuse many graphics screens in color, and are controlled by keyboard-or mouse-operated menus; however, they can also be controlledby command-line parameters for standard or repetitive input.     

Prostaglandin biosynthesis by human decidual cells: effects of inflammatory mediators

There is substantial evidence that decidual activation, in association with infection, is linked with the onset of both preterm and term labor. We therefore undertook the present study to evaluate prostaglandin production and its potential regulation by inflammatory mediators in human decidual cells in primary monolayer culture. Upon attaining confluence, the cells were incubated with endotoxin, interleukin 1 alpha (IL1 alpha), interleukin 1 beta (IL1 beta); or tumor necrosis factor (TNF). Production of prostaglandin (PG) E2 and PGF2 alpha was determined using specific radioimmunoassays. Endotoxin and these cytokines all induced significant concentration-dependent increases in PGE2 and PGF2 alpha production. Our results suggest that term human decidual cells are responsive to endotoxin and cytokines and that generation of these substances in the decidua or nearby (eg. in response to infection) will lead to increased prostaglandin production and uterine contractions.     

Novobiocin-resistance sequences from the novobiocin-producing strain Streptomyces niveus

Two distinct DNA sequences expressing novobiocin resistance in Streptomyces lividans were cloned from the novobiocin-producing species Streptomyces niveus. Clone pGL101 (5kb) conferred resistance to 50 micrograms ml-1 novobiocin, whereas clones pGL102 and pGL103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150 micrograms ml-1. The cloned inserts from pGL101 and pGL103 failed to hybridize with each other or with the cloned novobiocin-resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin-producing species S. niveus and S. sphaeroides but no hybridization (pGL103) or very weak hybridization (pGL101) was detected with DNA from the non-producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin-resistance determinants with the pGL101 and pGL103 cloned sequences specific for novobiocin-producing strains of Streptomyces.     

Diagnosis of abdominal masses with percutaneous biopsy guided by ultrasound.

OBJECTIVE--To assess the accuracy and safety of percutaneous biopsy of abdominal masses guided by ultrasound. DESIGN--Prospective study. SETTING--Combined gastroenterology service, Scarborough Hospital. PATIENTS--108 Consecutive patients identified as having a discrete mass on diagnostic ultrasound examination of the abdomen. INTERVENTION--A sample of tissue was obtained with an aseptic technique under local anaesthesia: an 18 steel wire gauge needle (Tru-Cut) was mounted in a spring loaded firing device (Biopty gun) that was advanced under simultaneous ultrasound scanning, permitting precise localisation of the target organ. MAIN OUTCOME MEASURE--Results of histological examination of tissue specimens. RESULTS--Biopsy failed in four patients. Adequate histological specimens were obtained in 104 patients with masses in the liver (31), pancreas (37), kidney (10), and adrenal glands (six) and in 20 undiagnosed abdominal and retroperitoneal masses. Follow up was until death or confirmation of the diagnosis. Three complications but no deaths occurred. Malignancy was suspected in 84 patients before biopsy. This was confirmed in 70 patients, in 26 of whom confirmation of dissemination obviated the need for further investigation. In 10 patients biopsy indicated a previously unsuspected primary tumour, and in 12 it showed only a benign lesion. Among 24 patients considered to have benign disease biopsy showed an unsuspected neoplasm in seven. Use of biopsy thus had a major effect on clinical management in 55 patients. Four false negative but no false positive diagnoses resulted from the procedure. CONCLUSION--Percutaneous biopsy of abdominal and retroperitoneal masses under ultrasound guidance is a safe and accurate method of obtaining a histological diagnosis. The results obtained have a considerable effect on clinical management.