The effect of hemoglobin ligands on the kinetics of human hemoglobin A1c formation

HbA1c is the most prevalent of the minor human hemoglobins. It is formed by the nonenzymatic addition of glucose to the alpha-amino group of the beta chain by an initial condensation reaction and a subsequent intermolecular Amadori rearrangement. We have developed a method of analysis which utilizes high performance liquid chromatography to follow the formation of HbA1c and greatly simplifies the determination of the kinetic parameters associated with this reaction. This has allowed us to study the effects of several Hb ligands, including the hydrogen ion, on the kinetics of this glycosylation reaction. Both the initial condensation reaction and the subsequent rearrangement are shown to exhibit acid catalysis, but the rate of the condensation step is limited by the extent of protonation of the alpha-amino group. The variation in kinetic parameters as a function of hydrogen ion concentration has allowed us to determine the probable reaction mechanism of HbA1c formation by comparison to previously reported model systems of Schiff base formation and Amadori rearrangement. The formation of pre-HbA1c from deoxy-Hb shows an increased forward rate when compared to oxy-Hb. The presence of physiologic concentrations of CO2 causes a proportional decrease in both k1 and k-1. 2,3-Diphosphoglycerate causes a significant increase in the keq of the formation reaction. The effects of CO and the substitution of L-glucose for D-glucose are not significant.     

Aromatization of androstenedione by normal and neoplastic endometrium of the uterus

The ability of human uterine endometrium to aromatize androstenedione to estrogens was investigated using 10 normal and neoplastic tissues. Normal and neoplastic endometrial homogenates were incubated with [6,7-3H]androstenedione (A) and NADPH. Estrone (E1) and estradiol (E2) were subsequently isolated in amounts ranging from 0-17600 fmol/h/g and 0-377 fmol/h/g, respectively, from the incubates after purifications by using Bio-Rad AG1-X2 column, thin layer chromatographies and co-crystallization. The conversion of A to E1 and E2 was significantly higher in neoplastic tissues.     

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.     

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.