Survival, mutagenesis, and host cell reactivation in a Chinese hamster ovary cell ERCC1 knock-out mutant

Positive selection-negative selection gene targeting was usedto disrupt the nucleotide excision repair gene ERCC1 in a Chinesehamster ovary cell line, CHO-K1. Southern and Northern analysisshowed that a cell clone isolated by this targeting approach,CHO-7-27, had an ERCC1 gene structure consistent with targeteddisruption of ERCC1 exon V, and did not express ERCC1 mRNA.CHO-7-27 was further characterized with respect to UV and mitomycinC sensitivities, and was shown to exhibit severe mutagen sensitivityphenotypes consistent with those of other CHO cell ERCC1 mutants.Mutation frequency experiments showed that CHO-7-27 was UV-hypermutableat the hypoxanthine-guanine phosphoribosyltransferase locus.Experiments assessing host cell reactivation of viral DNA synthesisfor UV-irradiated adenovirus showed that CHO7-27 exhibited aseverely deficient HCR phenotype similar to that of UV20 cells.Our results demonstrate that CHOK1 cells are hemizygous forthe ERCC1 gene, and show that the comparatively mild mutagensensitivities and lack of severely deficient HCR phenotypesof conventionally derived CHO-K1 ERCC1 mutants, in contrastto the severe phenotypes of CHO-AA8-derived mutants, are notdue to any intrinsic genetic differences between CHO-K1 andCHO-AA8 parental cell lines. ⁴To whom correspondence should be addressed     

Phosphorylated Mr 32,000 dopamine- and cAMP-regulated phosphoprotein inhibits Na+,K(+)-ATPase activity in renal tubule cells.

Dopamine inhibits Na+,K(+)-ATPase activity in several renal tubule segments and thereby regulates urinary Na+ excretion. We now show that a phosphopeptide of 31 amino acids, corresponding to residues 8-38 of the protein phosphatase inhibitor DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000), mimics the inhibitory action of dopamine on Na+,K(+)-ATPase activity in renal tubule cells from the ascending limb of the loop of Henle. The dephosphorylated form of the peptide is ineffective. The results indicate that dopamine acts through a protein phosphorylation pathway to regulate the activity of an ion pump. In addition, the data suggest that inhibition of protein phosphatase 1 by phophorylated DARPP-32 is a component of the mechanism by which dopamine regulates urinary Na+ excretion.     

Pulpal reactions to glutaraldehyde and paraformaldehyde pulpotomy dressings in monkey primary teeth

Abstract Pulpotomy was performed in primary teeth of 4 vervet monkeys to compare the effect of an experimental dressing containing glutaraldehyde (GL) to a similar formula with paraformaldehyde (PF). Forty teeth were examined histologically 2 weeks to 5 months post-operatively. In the PF group, calcifications were more frequently observed and the central core of tissue in the root canal was heavily laden with debris. Pathologic apical resorption and lesions were more frequent. The pulp in the apical third of the root canal was usually uninflamed in the GL group and peripheral calcifications were seldom observed in it. Reaction to GL seemed, therefore, more favorable clinically. Defects were not observed in the permanent teeth that erupted following shedding or extraction of the experimental primary ones of either group.     

Formulation parameters affecting the preparation and properties of microencapsulated ion-exchange resins containing theophylline

A method of microencapsulating theophylline ion-exchange resins with ethylcellulose was developed to produce smooth and uniform coats which were predominantly mononucleated. This was achieved by controlling the amount of ethylcellulose and the particle size, and through the use of a protective colloid, polyisobutylene. The rate of release of theophylline was influenced by the ion-exchange resin crosslinking, the amount of ethylcellulose, and the smoothness of the coat. Mesh size and polyisobutylene did not appear to affect the rate in a regular manner. It was found that the release rate from coated resins with low crosslinking followed a logarithmic plot, indicating membrane-controlled release, whereas coated resins with high crosslinking fitted a t1/2 plot, suggesting particle diffusion control.     

Effects of the antimigraine compound zolmitriptan ('Zomig') on psychomotor performance alone and in combination with diazepam in healthy volunteers

Zolmitriptan (Zomig^TM) is a 5HT_1B/1D agonist which has the ability to cross the intact blood-brain barrier to access central as well as peripheral receptors. Because of the potential for central nervous system side effects, this randomized, double-blind, placebo-controlled, 6-period crossover study evaluated the effects of 2.5 and 5 mg doses of zolmitriptan on psychomotor performance and investigated any pharmacodynamic or pharmacokinetic interaction with diazepam. Twelve healthy volunteers received the following "treatments" as single doses: zolmitriptan 2.5 mg, zolmitriptan 5 mg, diazepam 10 mg, zolmitriptan 2.5 mg+diazepam 10 mg, zolmitriptan 5 mg+diazepam 10 mg and placebo. Pre-dose and at 1, 4, 8, and 24 h post-dose, the following validated battery of psychomotor tests was performed: Bond-Lader visual analogue scales (calmness, contentedness, and alertness factors), critical flicker fusion test, choice reaction time (recognition, motor, and total reaction times), finger-tapping test, number cancellation test and digit symbol substitution test. Plasma concentrations of zolmitriptan, its active metabolite, and diazepam and its active metabolites were measured at the same timepoints. Zolmitriptan 2.5 and 5 mg had no effect on psychomotor function when given alone. In contrast, diazepam 10 mg had profound effects, consistent with its sedative properties, but there was no synergism on concomitant administration of either dose of zolmitriptan. Plasma concentrations of zolmitriptan, diazepam, and their respective active metabolites were similar when the two drugs were given alone or in combination.     

PCPP-260, a Purkinje cell-specific cyclic AMP-regulated membrane phosphoprotein of Mr 260,000

The present study reports the existence of Purkinje cell-specific phosphoprotein, Mr 260,000 (PCPP-260), a neuronal membrane phosphoprotein, in cerebellar Purkinje cells. PCPP-260, which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has an apparent molecular mass of 260,000 Da, has been found to be phosphorylated in particulate preparations by endogenous or added exogenous cyclic AMP-dependent protein kinase, but not by cyclic GMP-dependent, calcium/calmodulin-dependent or calcium/phospholipid-dependent protein kinases. The protein has been found in high concentrations in all mammalian cerebella so far analyzed, including human cerebellum. One-and two-dimensional electrophoretic and peptide mapping analyses of proteins in other brain regions show that a closely related 265,000 Da phosphoprotein also exists, albeit in low concentrations, outside the cerebellum. Analysis of cerebella from mutant mice, deficient in either Purkinje cells or in granule cells, indicates that PCPP-260 within the cerebellum is restricted to Purkinje cells. Furthermore, subcellular fractionation of rat cerebella indicates that the protein is an integral membrane protein. The cAMP-regulated phosphorylation of PCPP-260 is presumably involved in membrane functions important to Purkinje cells.     

Microphotometry in immunofluorescence

A specially sensitive version of the Reichert microphotometer provides a means of obtaining accurate measurements of the emission of immunofluorescent microscopical preparations. The fluorescence of single particles down to 0·8 μ in diameter may be measured with all background excluded. The photometer has permitted determinations of titre of antisera, assessment of the validity of conventional visual estimation of fluorescence, and comparative studies of fluorescence intensity and fading under a variety of conditions. Preliminary studies by double immunofluorescent staining of two distinct antigenic constituents of cells which might be present either separately or together, suggest future value in investigations of cell differentiation and dedifferentiation.     

Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor

Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.     

Rhodamine as a fluorescent probe of lymphocyte activation.

Fresh rat and mouse lymphoid cells have been labelled by stable linkage with tetramethylrhodamine isothiocyanate (TMRITC). A change in intensity, either an increase or decrease of the fluorescent emission of the cells, detected by microfluorimetry, was induced by mitogen stimulation or the mixed lymphocyte reaction. The change in fluorescence was observed within 3 h of mitogen stimulation and within 0.5 h in the mixed lymphocyte test. These early cellular responses were detectable consistently whether the labelling was done before or after mitogen stimulation; post-labelling only was studied in the mixed lymphocyte reaction. The method should provide a time-saving practical procedure for early detection of the lymphoid cell responses and would readily lend itself to flow cytofluorimetry for possible routine diagnostic use.     

Fluorescence polarization assay by flow cytometry

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.