Properties of the selenium-containing moiety of nicotinic acid hydroxylase from Clostridium barkeri

The nicotinic acid hydroxylase from Clostridium barkeri is a selenoenzyme, as evidenced by the copurification of selenium with enzyme activity. This conclusion is supported by data showing a 23-fold increase in nicotinic acid hydroxylase activity when C. barkeri was cultured in media supplemented with selenium. A labile, selenium-containing compound was released from the native protein by treatment with either chaotropic agents and heat or by heating alone. A stable selenium compound was formed when the enzyme was alkylated prior to denaturation. This compound had the same chromatographic properties as dialykyl selenide in a number of systems. The formation of dialkyl selenide upon alkylation is not consistent with the selenium moiety being selenocysteine. Thus, nicotinic acid hydroxylase represents a new type of selenoenzyme.     

Synthesis of phosphorylated 3,4-branched trisaccharides corresponding to LPS inner core structures of Neisseria meningitidis and Haemophilus influenzae

2-(N-Benzyloxycarbonyl)aminoethyl 7-O-acetyl-6-O-allyl-2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-l-glycero-α-d-manno-heptopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl-(1→4)]-6,7-di-O-acetyl-2-O-benzyl-l-glycero-α-d-manno-heptopyranoside, a spacer-equipped protected derivative of the common 3,4-branched diheptoside trisaccharide structure of the lipopolysaccharide core of Neisseria meningitidis and Haemophilus influenzae has been synthesized. The protecting group pattern installed allows regioselective introduction of phosphoethanolamine residues in the 3- and 6-position of the second heptose unit in accordance with native structures. From this intermediate the 3-and 6-monophosphoethanolamine as well as the non-phosphorylated deprotected trisaccharides have been synthesized to be used in evaluation of antibody binding specificity and in investigation of the substrate specificity of glycosyl transferases involved in the biosynthesis of LPS core structures.     

Absent aldosterone response to metoclopramide in patients with high spinal cord transection: evidence that metoclopramide stimulates aldosterone secretion through central pathways

This study evaluates dopaminergic regulation of aldosterone secretion in 6 patients with high spinal cord transections. Administration of the dopamine antagonist metoclopramide resulted in a marked rise in plasma aldosterone and 18-hydroxycorticosterone levels in 12 normal individuals, but no change in plasma levels of these zona glomerulosa corticosteroid products in spinal cord patients. Spinal cord transected patients also did not have the rise in plasma renin activity that was observed in normals following metoclopramide administration. Basal levels of aldosterone, 18 hydroxycorticosterone, corticosterone and renin activity as well as the aldosterone responses to graded dose infusion of adrenocorticotropin were similar in the spinal cord patients and the normals. These data suggest that dopaminergic regulation of adrenal zona glomerulosa corticosteroid and renal renin secretion is absent in patients with high spinal cord transections, suggesting that intact neural pathways from the central nervous system are necessary for metoclopramide stimulation of aldosterone and renin secretion in men. Since basal plasma aldosterone levels were normal in spinal cord transected patients, it appears that the absence of dopaminergic control does not result in elevated secretion.     

D-2 dopamine receptors in the frontal cortex of rat and human

D-2 dopamine receptors and serotonin receptors in the frontal cortex of rat and human were labelled with 3H-spiroperidol. The D-2 receptors were then distinguished in 4 ways. Dissociation of spiroperidol was biphasic, indicating two populations of sites. Cinanserin in competition with 3H-spiroperidol exhibited high (75%) and low (25%) affinity sites. Dopamine and LY 141865 in competition with 1.25 nM 3H-spiroperidol exhibited high (20-25%) and low (80-75%) affinity sites in the absence of cinanserin, while in the presence of 300 nM cinanserin only the high affinity sites remained. Lesioning of the dopaminergic meso-cortical pathway increased the number of cinanserin-resistant sites by 26%. Thus 3H-spiroperidol binding in the presence of cinanserin can be used to selectively label D-2 receptors in the frontal cortex.     

Characterization of beta-lactamase from Mycobacterium butyricum ATCC 19979

beta-lactamase has been purified to a homogeneous state from Mycobacterium butyricum ATCC 19979. The molecular weight (Mr = 29,000) and the isoelectric point (4,0) of the enzyme have been determined. The enzyme showed both penicillinase and cephalosporinase activity, but had relatively more of the former. With respect to substrate-profile the enzyme resembled the plasmid specified TEM-type beta-lactamases commonly encountered in Gram-negative bacteria. The enzyme was insensitive to p-chloromercuribenzoate, sodium chloride, or iodine inhibition.     

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.