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.     

Molecular control of rabbit follicular testosterone production. Role of protein and RNA after stimulation with luteinizing hormone.

The time and dose dependence of the relationship between uptake of labelled precursors into protein and RNA and production of testosterone by rabbit follicles was examined. Although testosterone production was stimulated by luteinizing hormone at concentrations between 0.1 and 10 microgram/ml, the uptake of [3H]leucine into protein was significant only when the concentration of luteinizing hormone was greater than 2.5 microgram/ml. Increased production of testosterone was observed within 15 min of stimulation with luteinizing hormone whereas uptake of [3H]leucine was only significant at 90 min. Puromycin (40 microgram/ml) and cycloheximide (10 microgram/ml) in the presence of luteinizing hormone inhibited the synthesis of both testosterone and protein. However, lower concentrations of puromycin (0.1, 1 and 10 microgram/ml) and cycloheximide (1 microgram/ml) had no effect on luteinizing hormone-induced testosterone production but significantly inhibited protein synthesis by 58, 37, 31 and 71%, respectively. Actinomycin D (20, 80 and 160 microgram/ml) alone and in combination with 5 microgram luteinizing hormone/ml severely inhibited uptake of [3H]uridine into RNA without affecting testosterone production. However, with 1 microgram actinomycin/ml, testosterone production was significantly (P less than 0.01) greater than in the presence of luteinizing hormone alone. These results cast doubt on the obligatory role of RNA and protein synthesis in rabbit ovarian follicular steroidogenesis.     

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

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.     

On the Rotation of Canonical Solutions

Some points of CLIFF /KRUS 's important rotation procedure are criticized. This result in the definition of simple structure of canonical solutions, the use of STEWART /LOVE 's redundancy index as a measure of common variance, and two new rotation procedures (HAKSTIAN 's modified varimax rotation, separate rotation of both sets). The objects of rotation should be the (intraset) loadings.     

Properties of purified colchicine-binding protein from a cultured carrot cell extract

Colchicine-binding protein (CBP) was purified from a cultured carrot cell extract by DEAE-Sephacel, phosphocellulose and Sephadex G200 column chromatographies. The purified CBP separated into three bands on SDS-polyacrylamide gel electrophoresis. One of them reacted with a monoclonal antibody against chick brain alpha-tubulin and the other two with that against beta-tubulin. Colchicine-binding activity of the purified protein was enhanced by tartrate and inhibited little by an excess of podophyllotoxin. It decayed following first order kinetics, but was more stable than the CBP in the crude extract. The binding constant of the purified CBP for colchicine was 0.57 microM-1 and the number of binding sites of colchicine per mg protein was about 2 nmol. This binding constant is about ten times lower than that of porcine brain tubulin under identical conditions.     

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.