Kinetics of dark repair inhibition in ultraviolet damaged DNA by substituted quinolines

The effects of chloroquine on the kinetics of the dark repair process were investigated by employing a cellular system. Lineweaver-Burke plots and Dixon plots of chloroquine inhibition, respectively, showed that there was an increase in slope with increasing concentration of inhibitor which followed an enzyme-like pattern. These results are consistent with a model for excision-repair in which chloroquine may block the excision repair pathway.     

Protein degradation during the differentiation of eukaryotic cells: studies on the sporulation of Saccharomyces cerevisiae and on the formation of the neuromuscular junction in the chick embryo.

The role of protein degradation in cell and tissue differentiation has been investigated during the sporulation of Saccharomyces cerevisiae and during endplate formation in developing avian muscle. The results suggest that a variety of proteolytic processes as enzyme inactivation, degradation of mitochondrial membrane constituents and removal of embryonic cell surface proteins exert stringent controls over the sequence of differentiation in eukaryotic cells.     

Formation of a barrier by brain microvessel endothelial cells in culture

Endothelial cells (EC) isolated from bovine brain microvessels produce a continuous monolayer when grown in primary culture. The EC are joined together by tight junctions and contain few pinocytotic vesicles. Horseradish peroxidase (HRP) is unable to penetrate this in vitro barrier system. Exposure of the cells to 1.6 M arabinose produces a reversible separation of the tight junctions with penetration of HRP across the monolayer in a pattern similar to that observed in animals after infusion of hyperosmotic solutions into the carotid artery. The behavior of brain microvascular cells in culture suggest that they retain properties important to the formation of the blood-brain barrier.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Symmetric lac operator derivatives: effects of half-operator sequence and spacing on repressor affinity

We have analyzed lac repressor binding in vivo and in vitro to several symmetric lac operator sequences. Two features of the operator appear to be important for repressor binding: sequence, both of the operator and of its extended regions, and the spacing of the operator halves. Host mutations that alter DNA superhelical density (topA, gyrB) did not change the relative affinity of cloned symmetric operator sequences for repressor. Analysis by dimethylsulfate methylation and DNaseI digestion of repressor-operator complexes indicated that repressor makes symmetric contacts with the symmetric operator, in contrast to its contacts with the two halves of the natural operator.     

The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.     

Agonist pharmacology of neonatal and adult glycine receptor alpha subunits: identification of amino acid residues involved in taurine activation.

The inhibitory glycine receptor (GlyR) is a pentameric chloride channel protein which mediates postsynaptic inhibition in the mammalian central nervous system. In spinal cord, different GlyR isoforms originate from the sequential expression of developmentally regulated variants of the ligand binding alpha subunit. Here, neonatal alpha 2 and adult alpha 1 subunits are shown to generate GlyRs with distinct agonist activation profiles upon heterologous expression in Xenopus oocytes. Whereas alpha 1 receptors are efficiently gated by beta-alanine and taurine, alpha 2 GlyRs show only a low relative response to these agonists, which also display a reduced sensitivity to inhibition by the glycinergic antagonist strychnine. Construction of an alpha 2/alpha 1 subunit chimera and site-directed mutagenesis of the extracellular region of the alpha 1 sequence identified amino acid positions 111 and 212 as important determinants of taurine activation. Our results indicate the existence of distinct subsites for agonists on alpha 1 and alpha 2 GlyRs and suggest that the ligand binding pocket of these receptor proteins is formed from discontinuous domains of their extracellular region.     

Transport of ions across the blood-brain barrier

Capillaries in the brain are formed by a uniquely specialized endothelial cell that regulates the movement of substances between blood and brain. Although they provide an impermeable barrier to some solutes, brain capillary endothelial cells facilitate the transcapillary exchange of others. In addition, they contain specific enzymes that contribute to a metabolic blood-brain barrier by limiting the movement of compounds such as neurotransmitters across the capillary wall. Studies of sodium and potassium transport by brain capillaries indicate that the endothelial cell contains distinct types of ion transport systems on the two sides of the capillary wall, i.e., the luminal and antiluminal membranes of the endothelial cell. As a result, specific solutes can be pumped across the capillary against an electrochemical gradient. These transport systems are likely to play a role in the active secretion of fluid from blood to brain and in maintaining a constant concentration of ions in the brain's interstitial fluid. In this way, the brain capillary endothelium is structurally and functionally related to an epithelium.