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.     

Role of human fetal ependyma.

Fetal ependyma is an active secretory structure for the programming of developmental events, including the arrest of neuronogenesis, the guidance of axonal growth cones, motor neuron differentiation, and probably also the maintenance and transformation of radial glial cells that guide migratory neuroblasts. The floor plate, induced by the notochord, is the first part of the neuroepithelium to differentiate. It establishes polarity and growth gradients of the neural tube and has immunohistochemical features that differ from all other regions of the ependyma. The dorsal and ventral median septa, formed by floor and roof plate ependymal processes, prevent aberrant decussations of developing long tracts, but permit the passage of commissural axons. Fetal ependyma synthesizes several intermediate filament proteins absent from mature ependymal cells, although some are also expressed in undifferentiated neuroepithelial cells. Fetal ependyma also produces diffusible molecules, such as neural cell adhesion molecule, proteoglycans, nerve growth factor, and S-100 protein, all in specific temporal and spatial distributions. Maturation of the ependyma is not complete until the postnatal period. An abnormal fetal ependyma may play a primary role in the pathogenesis of some cerebral malformations, such as lissencephaly/pachygyria and holoprosencephaly.     

Watershed infarcts in the fetal and neonatal brainstem. An aetiology of central hypoventilation, dysphagia, M?ibius syndrome and micrognathia.

Watershed zone infarcts of the human cerebral cortex at the overlapping junctions of the anterior and middle cerebral arterial territories are well known. Another watershed zone exists in the brainstem tegmentum, between the terminal perfusion zones of the paramedian penetrating and long circumferential arteries, which are paired segmental vessels arising from the basilar artery. The vertebrobasilar circulation achieves its mature configuration and caudorostral flow by 9 weeks gestation. Systemic hypotension and other conditions of reduced basilar perfusion in the fetus, either early or late in gestation, may result in symmetrical longitudinal columns of infarction in the midbrain and tegmentum of the pons and medulla oblongata and laminar necrosis of the midbrain tectum. Within this zone are cranial nerve nuclei III-XII, the nucleus and tractus solitarius or central pneumotaxic center, as well as the nucleus ambiguus and other somatic motor nuclei that subserve muscles of swallowing, mastication and tongue movement. Watershed infarcts in the human fetal and neonatal brainstem are clinically expressed as multiple cranial neuropathies, failure of central respiratory drive and apnea, dysphagia and aspiration, M?bius syndrome and Pierre Robin sequence. MRI is sometimes helpful, but most of the involved neuroanatomical structures are beneath the resolution of present imaging techniques, and the diagnosis during life depends upon clinical neurological examination of the neonate, sometimes supported by evoked potential studies. Postmortem examination confirms the diagnosis and dates the lesions, but also contributes to better understand transient or persistent vascular insufficiencies in the fetal and neonatal brainstem.     

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.     

Do the corticospinal and corticobulbar tracts mediate functions in the human newborn?

Unlike the numerous dispersed bulbospinal pathways that are already well myelinated at term, the more compact corticospinal and corticobulbar tracts are only beginning their myelination cycle in late gestation and do not complete it until two years of age. During this same period, these pathways also develop extensive ramification of terminal axonal segments, growth of collateral axons, and proliferation of synapses. Despite their immaturity in the full-term human newborn, several proposed functions may be attributed to the descending pathways from the neonatal cerebral cortex: a) a contribution to the differential development of passive muscle tone and resting postures; in general they function as an antagonist to the "subcorticospinal pathways" in mediating proximal flexion and distal extension, except for the rubrospinal tract which is probably synergistic with the corticospinal tract; b) enhancement of tactile reflexes originating in the brainstem and spinal cord, including suck and swallow; c) relay of epileptic activity of cortical origin; d) inhibition of complex stereotyped motor reflexes including many phenomena formerly termed "subtle seizures"; e) a possible influence on muscle maturation, particularly in relaying cerebellar impulses that modify the histochemical differentiation of myofibres. However, the bulbospinal tracts are probably more influential on muscle development. The corticospinal and corticobulbar tracts subserve different needs in the newborn than at older ages, but are functionally important pathways even at birth.