Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.     

Determination of the Activity of Immobilized Biocatalysts in the Case of Poorly Soluble Substrates: The Example of D-5Phenylhydantoinase

The present communication describes the determination of activity of immobilized biocatalysts, from progress curves, in the case of a poorly soluble substrate. Computer simulation is used for the comparison of five well known methods for the determination of initial velocity of the reaction with the modified method of smoothing experimental data by cubic spline. The computer simulation data show that, in the case of poorly soluble substrate, it is expedient to use the method of data linearization in the co-ordinate system p/t versus p and method of splines.     

Significance of the smell of a conspecific for the spatial distribution of the common shrew Sorex araneus L.

The significance of smell marks of conspecifics for the spatial distribution of common shrews was studied. The existence of two groups of individuals, which differ in their reaction to the smell of a conspecific, was shown. Individuals with different reactions were shown to have reliable differences in the sizes of the areas visited by them, the mutual location of their plots, and the percent of activity combined with the activity of the neighbor. The significance of such differences in reactions for the formation of the social system of shrews is discussed.     

Inhibitory effect of carboxylic acid group on hERG binding

Drug-induced QT prolongation arising from drugs' blocking of hERG channel activity presents significant challenges in drug development. Many, but not all, of our benzamidine-containing factor Xa inhibitors were found to have high hERG binding propensity. However, incorporation of a carboxylic acid group into these benzamidine molecules generally leads to hERG inactive compounds regardless where the carboxyl group is tethered within the molecules. The inhibitory effect of a carboxylic acid group on hERG binding has also been observed in many series of diverse structural scaffolds (including non-amidines). These findings suggest that the negatively charged carboxylate group causes unfavorable interaction within hERG channel binding cavity by electrostatic interaction.     

A common polygenic basis for quinine and PROP avoidance in mice

Inbred strains of mice (Mus musculus) differ greatly in ability to taste various bitter compounds. For some compounds, the differences result from allelic variation at a single locus. However, segregation patterns incompatible with monogenic inheritance have been found for quinine avoidance. The Soa bitter sensitivity locus exerts some influence on this phenotype, but an unknown number of other loci also contribute. Relative avoidance patterns for quinine sulfate in panels of naive inbred strains resembled avoidance patterns for 6-n-propyl-2- thiouracil (PROP), suggesting a common genetic basis. In particular, C57BL/6J mice strongly avoided both 0.1 mM quinine sulfate and 1 mM PROP in two-bottle preference tests, whereas C3H/HeJ mice were indifferent to both. Therefore, 12 BXH/Ty recombinant inbred strains, derived from these strains, were tested with both solutions to begin identification of the unknown bitter loci. Naive mice were tested for four consecutive days with each compound (order counterbalanced). Some BXH/Ty strain means resembled those of the parent strains, but others were intermediate. This indicated recombination among loci affecting avoidance, and therefore polygenic inheritance. The strain means were highly correlated across compounds (r = 0.98), suggesting that the same polygenes controlled both phenotypes. The BXH/Ty means for both compounds were then compared with the strain genotypes at 212 chromosome position markers distributed throughout the genome. Eight markers on five chromosomes (3, 6, 7, 8 and 9) yielded significant correlations. Six of the markers were correlated with both phenotypes, again suggesting common polygenic inheritance. The marker with the highest correlation was Prp, tightly linked to Soa on chromosome 6. The correlated marker regions likely contain quantitative trait loci affecting bitter avoidance. The phenotypic similarity of PROP to quinine, rather than to phenylthiourea, apparently stemming from a common polygenic basis, indicates a difference between mice and humans in gustatory organization related to bitters.      

Role of Nitric Oxide on Motor Behavior

The present review paper describes results indicating the influence of nitric oxide (NO) on motor control. Our last studies showed that systemic injections of low doses of inhibitors of NO synthase (NOS), the enzyme responsible for NO formation, induce anxiolytic effects in the elevated plus maze whereas higher doses decrease maze exploration. Also, NOS inhibitors decrease locomotion and rearing in an open field arena.These results may involve motor effects of this compounds, since inhibitors of NOS, NG-nitro-L-arginine (L-NOARG), N^G-nitro-L-arginine methylester (L-NAME), N^G-monomethyl-L-arginine (L-NMMA), and 7-Nitroindazole (7-NIO), induced catalepsy in mice. This effect was also found in rats after systemic, intracebroventricular or intrastriatal administration.Acute administration of L-NOARG has an additive cataleptic effect with haloperidol, a dopamine D2 antagonist. The catalepsy is also potentiated by WAY 100135 (5-HT1a receptor antagonist), ketanserin (5HT2a and alfa1 adrenergic receptor antagonist), and ritanserin (5-HT2a and 5HT2c receptor antagonist). Atropine sulfate and biperiden, antimuscarinic drugs, block L-NOARG-induced catalepsy in mice.L-NOARG subchronic administration in mice induces rapid tolerance (3 days) to its cataleptic effects. It also produces cross-tolerance to haloperidol-induced catalepsy. After subchronic L-NOARG treatment there is an increase in the density NADPH-d positive neurons in the dorsal part of nucleus caudate-putamen, nucleus accumbens, and tegmental pedunculupontinus nucleus. In contrast, this treatment decreases NADPH-d neuronal number in the substantia nigra compacta.Considering these results we suggest that (i) NO may modulate motor behavior, probably by interfering with dopaminergic, serotonergic, and cholinergic neurotransmission in the striatum; (ii) Subchronic NO synthesis inhibition induces plastic changes in NO-producing neurons in brain areas related to motor control and causes cross-tolerance to the cataleptic effect of haloperidol, raising the possibility that such treatments could decrease motor side effects associated with antipsychotic medications.Finally, recent studies using experimental Parkinsons disease models suggest an interaction between NO system and neurodegenerative processes in the nigrostriatal pathway. It provides evidence of a protective role of NO. Together, our results indicate that NO may be a key participant on physiological and pathophysiological processes in the nigrostriatal system.     

IL-4 (B cell stimulatory factor 1) overcomes Fc gamma receptor-mediated inhibition of mouse B lymphocyte proliferation without affecting inhibition of c-myc mRNA induction

Mouse B cells are stimulated to proliferate by Fab'2 fragments of rabbit anti-mouse Ig antibodies. Proliferation is inhibited, however, in the presence of IgG anti-mouse Ig. We have previously shown that this inhibition is mediated by binding of the IgG anti-Ig to receptors for Fc gamma R on B cells. This report describes conditions under which IgG anti-mu or anti-delta will induce proliferation despite Fc gamma R engagement. Culture supernatants of Con A-stimulated, Il-4-secreting Th cell lines, but not of Il-2-secreting Th cell lines, will co-stimulate with IgG anti-Ig to induce small B cells to incorporate [3H]TdR. This co-mitogenic activity is inhibitable by anti-IL-4 antibodies and can also be induced by Il-4 affinity purified from the T cell supernatants or by supernatants containing rIl-4. B cells precultured with Il-4 for 18 h, while still expressing normal levels of Fc gamma R, also proliferate to IgG anti-Ig. We have previously shown that Fc gamma R-mIg cross-linking will inhibit mIg-dependent increases in c-myc mRNA levels. We investigated whether Il-4 allows B cells to respond to IgG anti-Ig by elevating c-myc. The data show that Il-4 has little effect on c-myc mRNA levels in either IgG or Fab'2 anti-Ig-containing cultures.