Eryloside F, a novel penasterol disaccharide possessing potent thrombin receptor antagonist activity.

We report the discovery of Eryloside F, a novel disaccharide of the steroidal carboxylic acid penasterol, isolated from an extract of the marine sponge Erylus formosus. The compound is a potent thrombin receptor antagonist, and furthermore inhibits human platelet aggregation in vitro.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

Angiographic findings in patients undergoing catheterization for recurrent symptoms within 30 days of successful coronary intervention.

In 108 consecutive patients without abrupt vessel closure referred for repeat coronary angiography within 30 days of successful coronary intervention, 28 (26%) were found with restenosis at the treated site. None of the 27 patients who underwent stenting were found to have early restenosis; balloon angioplasty without stenting was the only independent predictor of early restenosis in patients with recurrent symptoms within 30 days of intervention.

The use of tailed octamer primers for cycle sequencing.

Studies have been carried out on the use of octamer oligonucleotides tailed with different base analogues as primers in cycle sequencing reactions. 5-Nitroindole tails improved the performance as primers of a number of octamers. A tail length of three or four 5-nitroindole residues significantly increased the sequencing signal intensity for almost all primers. The use of incomplete libraries of tailed octamer primers for primer walking is discussed.

On the number of automated timed manual performance trials to administer.

OBJECTIVE: To determine the number of trials needed to obtain satisfactory results when evaluating function in older subjects using the automated version of the Williams short (three-door) Timed Manual Performance (TMP) test. DESIGN: Administration of from one to five trials in succession on a given test date. SETTING: A Continuing Care Retirement Community (CCRC), assisted living centers, nursing homes, and a community residence, all located in central North Carolina. PARTICIPANTS: The subject population consisted of 182 older volunteers, aged 63 to 100 years. One hundred nineteen lived independently in a CCRC, 33 were assisted-living residents, 29 lived in nursing homes, and one lived independently in the community. Each subject was administered at least three trials in succession on his or her first test date; 23 of the independently living CCRC residents were given three trials on a second test date. The community-dwelling volunteer was administered from one to five trials on each of 26 test dates over an approximately 16-month period. MEASUREMENTS: The time it takes to perform five door opening and closing operations as measured by the three-door Cognatemp Automated Timed Manual Performance (ATMP) system. MAIN RESULTS: Average ATMP time for the subjects living independently decreased approximately 10% between the first two trials but negligibly between trials two and three. The more dependent groups continued to improve between trials two and three. The community-dwelling subject tended to improve in the first three or four trials and to decline by the fifth trial. CONCLUSIONS: It is recommended that two trials be administered and the best time used; if neither trial results in a time less than 10 seconds, one or two more trials should be administered. It is generally not necessary nor advantageous to administer more than four trials.

Increased anxiety of mice lacking the serotonin1A receptor.

Brain serotonin (5-HT) has been implicated in a number of physiological processes and pathological conditions. These effects are mediated by at least 14 different 5-HT receptors. We have inactivated the gene encoding the 5-HT1A receptor in mice and found that receptor-deficient animals have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation, behaviors consistent with an increased anxiety and stress response. Based on the role of the 5-HT1A receptor in the feedback regulation of the 5-HT system, we hypothesize that an increased serotonergic neurotransmission is responsible for the anxiety-like behavior of receptor-deficient animals. This view is consistent with earlier studies showing that pharmacological activation of the 5-HT system is anxiogenic in animal models and also in humans.

Iodine-131 in breast milk following therapy for thyroid carcinoma.

UNLABELLED: This study evaluates breast milk secretion of 131I following therapeutic administration of 4000 MBq of 131I-iodide during lactation. METHODS: Breast milk 131I activity concentration was measured over a 32-day period. Dosimetry calculations were undertaken to estimate the period for discontinuation of breast feeding and the equivalent dose to the breasts. RESULTS: To achieve an infant effective dose < 1 mSv and an infant thyroid dose < 10 mSv, breast feeding would need to be discontinued for at least 52 days. The estimated equivalent dose to the breasts was 1.6 Gy. CONCLUSION: It is suggested that 131I-iodide administration is not undertaken during lactation and that breast feeding is discontinued several days prior to administration.

Kinetic parameters for the generation of endothelins-1,-2 and -3 by human cathepsin E.

The specific conversion of human endothelin (ET) precursors big ET-1, big ET-2 and big ET-3 into their respective ET by cathepsin E was examined. Comparable pH optima were obtained for ET-1 and ET-2 generation, whereas effective conversion of big ET-3 into ET-3 necessitated a lower pH value. Determination of kinetic parameters (Km, kcat.) for all three conversions indicated that the precursors were efficiently bound by cathepsin E. The significance of the values obtained for the catalytic-centre activities and the effect of a specific inhibitor are discussed.

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.

Kidney-specific enzyme expression by human kidney cell lines generated through oncogene transfection.

The human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST-1i and STt-4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney-specific enzyme activity levels in 293, ST-1i, and STt-4i cells to determine their ability to exhibit kidney-specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (gamma-GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC-PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney-specific enzyme activities was assessed in response to several differentiation-inducing agents (adenosine, n-butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST-1i and STt-4i exhibit elevated levels of LAP, gamma-GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just gamma-GTP and maltase. Maltase and gamma-GTP enzyme activities in ST-1i and STt-4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST-1i and STt-4i are comparable to normal HEK cells in expression of kidney-specific enzymes and in responsiveness to differentiation-inducing agents, in spite of continued expression of SV40 oncogenes.

Bile leaks after simple cholecystectomy.

Drains after cholecystectomy are used commonly to avoid biliary leaks and subsequent peritonitis. Thirty-five patients who had had cholecystectomy without drainage underwent 99mTc-labelled dimethylphenylcarbamoylmethyliminodiacetic acid (HIDA) and ultrasound scans the morning after surgery. Biliary leaks detected by positive HIDA scans occurred in 11 patients. Subhepatic fluid collections were seen on 20 ultrasound scans. There was no relation between biliary leaks and subhepatic collections. Many of the collections were not seen on the HIDA scan, suggesting that they contained blood and not bile. Many of the patients with bile leaks showed no fluid collection. Clinical complications were few and evenly distributed between those with positive and negative scans. We conclude that bile leaks are not an occasional event but occur after 31 per cent of undrained cholecystectomies and that these bile leaks remain clinically unimportant.

ADP-ribosylation of thylakoid membrane polypeptides by cholera toxin is correlated with inhibition of thylakoid GTPase activity and protein phosphorylation.

Incubation of pea thylakoid membranes with [32P]-NAD+ in the presence of cholera toxin resulted in the [32P]-ADP-ribosylation of a 60 kDa thylakoid membrane polypeptide. When ATP was included in the incubation mixture, a 29 kDa polypeptide was also labelled. In the absence of electron transfer cofactors or inhibitors, the extent of labelling depended on whether the membranes were preincubated in the light or dark and also on the developmental stage of the leaves used for thylakoid isolation. Irrespective of the latter, the strongest labelling was observed when DCMU was present in the light. After pretreatment of the thylakoid membranes with cholera toxin plus NAD+ under the same conditions, light-stimulated GTPase activity and protein phosphorylation were inhibited. The extent of inhibition for both processes appeared to be correlated with the amount of [32P]-ADP-ribosylation found when [32P]-NAD+ was included in the pretreatment mixture. The data presented are fully consistent with the 60 and 29 kDa polypeptides functioning as thylakoid membrane associated guanine nucleotide binding regulatory proteins.

Radionuclide localisation of parathyroid abnormality.

There is much discussion about the ideal method for pre-operative localisation of parathyroid abnormality, ie, adenoma or hyperplasia, but as yet there is no simple and/or non-invasive screening method available. Different clinical centres have access to different imaging modalities and therefore use a variety of techniques ranging from ultrasound to direct venous sampling. This paper presents a version of a dual radionuclide computerised subtraction procedure, but the author wishes to point out that this technique is still undergoing research.