Stacking and hydrogen bonding interactions between phenylalanine and guanine nucleotide: crystal structure of L-phenylalanine-7-methylguanosine-5'-monophosphate complex

The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein.     

A human monoclonal antibody directed to blood group i antigen: heterohybridoma between human lymphocytes from regional lymph nodes of a lung cancer patient and mouse myeloma

A fusion of human lymphocytes released from regional lymph nodes of papillary adenocarcinoma of lung cancer with mouse myeloma P3-X63-Ag8-U1 cells resulted in a stable hybridoma-secreting human IgM antibody (NCC-1004) that reacts with a large proportion of squamous cell carcinomas of lung and esophagus as well as carcinoma of thyroid glands. However, the antibody also reacts with normal red blood cells, B lymphocytes, and a few other limited loci in normal tissues such as the basal cells of bronchial epithelium and the basal cell layer of stratified squamous epithelium, as well as endothelium and alveolar lining epithelium. The antigen defined by NCC-1004 has been characterized as blood group i antigen on the basis of the following results. The antibody preferentially agglutinates cord erythrocytes in contrast to adult erythrocytes. The agglutination was obvious at 4 degrees C, but diminished greatly at 37 degrees C, and was enhanced after sialidase treatment. The antibody specifically reacts with lacto-norhexaosylceramide (nLc6) and sialosyllacto-norhexaosylceramide (IV3NeuAcnLc6), but does not react with lacto-neotetraosylceramide (nLc4), sialosyllacto-neotetraosylceramide (IV3NeuAcnLc4), lacto-isooctaosylceramide (IV6Gal beta 1----4GlcNAcnLc6; I antigen), and other standard glycolipids so far tested. The properties of the antibody and its antigen are identical to those previously described for the i blood group system. Inasmuch as the hybridoma was established by hybridization of lymphocytes derived from regional lymph nodes of lung cancer, and the antigen was found in the patient's lung cancer tissue, the i antigen in lung cancer is probably recognized as a tumor-associated antigen by the host's immune cell system.     

Performance of hydrophobic chromatography in purification of alpha-amylase

Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors.     

Evidence for unique homologous peptide sequences around the glycosylated seryl and threonyl residues in polysialoglycoproteins isolated from the unfertilized eggs of the Pacific salmon Oncorhynchus keta

Structures of glycopeptides obtained by exhaustive Pronase digestion of high molecular weight (1.7 X 10(5)) salmon egg polysialoglycoprotein have been elucidated. Six principal glycopeptides isolated by gel chromatography and DEAE-Sephadex A-25 chromatography in the absence or presence of borate ion were analyzed for their carbohydrate and amino acid composition, as well as amino acid sequence, and found to be of two distinct types: glycotripeptides, Thr*-Ser*-Glu, and glycotetrapeptides, Thr*-Gly-Pro-Ser, where an asterisk indicates the amino acid residues to which either the Gal beta 1----3GalNAc or Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3GalNAc chain is attached. Their final yield corresponds to 64% of the original desialylated glycoprotein. In view of the simple amino acid composition of salmon egg polysialoglycoprotein (molar ratio Asp2Thr2Ser3Glu1Pro1Gly1Ala3) and the result of alkaline beta-elimination indicating three carbohydrate units linked to two of two threonine and one of three serine residues, a unique primary structure comprising repetitive sequences of the above two types of glycopeptides, which are interspersed by short nonglycosylated peptides consisting of alanine and aspartic acid, has been proposed for the core protein. The molecular secondary ion mass spectra of underivatized glycopeptides were used to obtain their structural information. The anomeric configuration of the proximal sugar-peptide linkages was proven to be alpha by proton nuclear magnetic resonance spectroscopy. This is the first systematic reported study of O-glycosidically linked glycopeptides by these instrumental methods.     

Stopped-flow rapid kinetics of anesthetic-induced phase transition in phospholipid vesicle membranes: nonlocalized fluctuation

Kinetics of the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine vesicle membrane was studied by the stopped-flow technique with turbidity detection. The observed change in turbidity was well characterized by a single-exponential decay curve with relaxation time in the millisecond range, although the existence of a faster process than the dead-time of the stopped-flow apparatus was inferred from the amplitude analysis. Relaxation times were determined as functions of 1-hexanol concentration and temperature just below phase transition. From the analysis based on the theories of nonequilibrium relaxation, it is concluded that the phase transition induced by 1-hexanol is governed by a nonlocalized fluctuation mechanism. The anesthetic-induced nonequilibrium state is unstable rather than metastable.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

Aspartate aminotransferase isozymes from rabbit liver. Purification and properties

Cytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase [EC 2.6.1.1] ) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.     

Differentiated pressor and sympathetic responses to dual brain stimulation: ventromedial hypothalamus versus locus coeruleus

Sensitivity of the ventromedial hypothalamus (VMH) to electrical stimulation was compared with that of the locus coeruleus (LC) in urethane-anesthetized rats. Based not only on current strengths required to elicit threshold effects, but also on magnitude of pressor responses to suprathreshold stimulation, the LC was consistently more sensitive than the VMH. Despite this greater pressor sensitivity, splanchnic nerve firing increased almost equally upon stimulation of either brain area. Similar comparisons made in other rats following bilateral adrenalectomy or pretreatment with a vasopressin antagonist showed no significant alteration of pressor and sympathetic responsiveness to stimulation of either the LC or the VMH. When frequency of neural firing was recorded from a lumbar sympathetic trunk instead of the splanchnic nerve, increases in sympathetic nerve activity produced by LC stimulation were significantly larger than those produced from the VMH. The results suggest that greater pressor sensitivity of the LC is due, at least in part, to stronger constriction in vascular beds innervated by the lumbar sympathetic chains.     

Effects of short-term treatment with calcium on the parathyroid gland of the rat,under particular consideration of the alteration of storage granules

Summary Short-term effects of CaCl₂-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl₂, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded (i) that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and (ii) that these processes may be accelerated during hypercalcemia.     

Mechanism of biliary secretion of membranous enzymes: bile acids are important factors for biliary occurrence of gamma-glutamyltransferase and other hydrolases

To elucidate the mechanism of biliary occurrence of gamma-glutamyl transferase [EC 2.3.2.2] and alkaline phosphatase [EC 3.1.3.1], the effect of bile acids on the biliary level of these enzymes was studied in vivo and in vitro. Following intravenous administration of taurocholate, the activities of both enzymes in rat bile increased markedly with a concomitant increase in the excretion of the bile acid. The biliary levels of these enzymes increased to reach a maximum at 10-20 min after administration of the bile acid and decreased thereafter. Right-side-out oriented rat liver canalicular membrane vesicles which localize gamma-glutamyltransferase, aminopeptidase M and alkaline phosphatase on their outer surface (Inoue, M., Kinne, R., Tran, T., Biempica, L., & Arias, I.M. (1983) J. Biol. Chem. 258, 5183-5188) were prepared. Upon incubation of the vesicles with either intact or heat-treated bile samples, the membranous enzymes were released from the vesicles in a time-dependent manner. Incubation of these vesicles with physiological concentrations of taurocholate also solubilized these enzymes from the membranes. Affinity chromatographic analysis on concanavalin A-Sepharose revealed that the transferase thus solubilized retained the hydrophobic domain responsible for anchoring the enzyme to membrane/lipid bilayers. These results indicate that bile acid(s) excreted into the bile canalicular lumen solubilized these enzymes from the apical membrane surface of the biliary tract cells by their detergent action.