Synthesis and in vitro bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor

A series of C-terminal deleted analogs of human growth hormone-releasing factor (hGRF) with either an amidated or a free carboxylic acid C-terminus were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. A gradual decrease of bioactivity down to 23% relative to hGRF was noted when the C-terminal amino acids were deleted to hGRF (1-34)OH. Further deletions, however, did not decrease the bioactivity because the potencies of the fragments, hGRF(1-31)NH2, (1-30)NH2 and (1-29)NH2 remained at about 50% of that of hGRF. Continual deletion of residues to hGRF(1-23)NH2, (1-22)NH2 and (1-21)NH2 still yielded bioactive fragments with full intrinsic activity despite very low potency. Only with the deletion down to hGRF(1-19)NH2 did the bioactivity completely disappear. Thus, together with the data published in a previous paper (1), the minimal biologically active core of hGRF with full intrinsic activity comprises the fragment (3-21).     

Separation of enantiomeric amines and acids using chiral ion-pair chromatography on porous graphitic carbon

The application of porous graphitic carbon as adsorbing phase for direct separation of enantiomeric acids and amines using chiral ion-pair chromatography is described. The enantiomeric amines were separated as diastereomeric ion pairs with N-benzyloxycarbonylglycyl-L -proline, N-benzyloxycarbonylglycylglycyl-L -proline, or captopril as the chiral counterion. High enantioselectivities were obtained for amines having a hydrogen bonding function in the vicinity of the asymmetrical carbon atom. Quinine was the chiral counterion used to separate the enantiomeric acids. The strongly UV-absorbing quinine improved detection of solutes having low UV-absorbing properties, e.g., (R,S)-2-chloropropionic acid, by “indirect detection.” Retention and stereoselectivity of enanticmeric acids were regulated by the quinine concentration and by the addition of carboxylic acids as well as polar modifiers, e.g., methanol and 2-propanol, to the mobile phase. © 1992 Wiley-Liss, Inc.     

Characterization of B and H blood-group active glycosphingolipids from human B erythrocyte membranes

Two blood group B active glycosphingolipids (B-I and B-II) previously isolated and highly purified from human B erythrocytes [21] were analysed first by degradation with α-D-galactosidase from coffee beans, α-L-fucosidase from bovine kidney and with 0,1 N trichloracetic acid; the native B-glycolipids as well as their degradation products were then investigated by methylation analysis with combined gas chromatography-mass spectrometry, by thin layer chromatography, twodimensional immunodiffusion and by the hemagglutination inhibition technique. Together with the results obtained by mass spectrometry of permethylated glycolipids [26] the following structures were elucidated: α-D-galactopyranosyl-(1 → 3)-[α-L-fucopyranosyl-(1 → 2)]-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide for the B-I glycosphingolipid and α-D-galactopyranosyl-(1 → 3)-[α-L-fucopyranosyl-(1 → 2)]-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide for the B-II glycosphingolipid. A H active glycolipid fraction from B erythrocytes further purified by thin layer chromatography was also investigated by methylation analysis. The pattern of its partially methylated alditol acetates was essentially the same as that of the α-galactosidase treated and permethylated B-I glycolipid. It also exhibited strongly precipitating and hemagglutination inhibiting H properties as well as the two α-galactosidase treated B-I and B-II glycosphingolipids. Based upon these data the following tentative structure was proposed: α-L-fucopyranosyl-(1 → 2)-D-galactopyranosyl-(1 → 4)-N-acetyl-D-glucosaminosyl-(1 → 3)-D-galactopyranosyl-(1 → 4)-D-glucopyranosyl-(1 → 1)-ceramide. Gas chromatographic analysis revealed sphingosine and lignoceric, nervonic and behenic acids to be the main components of the ceramide residues of the three glycosphingolipids. From the data presented the H active substance very probably can be regarded as the immediate precursor of the B-I glycosphingolipid from human B erythrocyte membranes.     

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.     

A stable carbon isotope study of dissolved inorganic carbon cycling in a softwater lake

The dissolved inorganic carbon (DIC) cycle in a softwater lake was studied using natural variations of the stable isotopes of carbon,¹²C and¹³C. During summer stratification there was a progressive decrease in epilimnion DIC concentration with a concomitant increase in ¹³C_DIC), due to preferential uptake of¹²C by phytoplankton and a change in the dominant CO₂ source from inflow andin situ oxidation to invasion from the atmosphere. There was an increase in hypolimnion DIC concentration throughout summer with a concomitant general decrease in ¹³C_DIC from oxidation of the isotopically light particulate organic carbon that sank down through the thermocline from the epilimnion.Mass balance calculations of DI¹²C and DI¹³C in the epilimnion for the summer (June 23–September 25) yield a mean rate of net conversion of DIC to organic carbon (C_org) of 430 ± 150 moles d^-1 (6.5 ± 1.8 m moles m^-2 d^-1. Net CO₂ invasion from the atmosphere was 420 ± 120 moles d^-1 (6.2 ± 1.8 m moles m^-2 d^-1) with an exchange coefficient of 0.6 ± 0.3m d^-1. These results imply that at least for the summer months the phytoplankton obtained about 90% of their carbon from atmosphere CO₂. About 50% of CO₂ invasion and conversion to C_org for the summer occurred during a two week interval in mid-summer.DIC concentration increased in the hypolimnion at a rate of 350 ± 70 moles DIC d^-1 during summer stratification. The amount of DIC added to the hypolimnion was equivalent to 75 ± 20% of net conversion of DIC to C_org in the euphotic zone over spring and summer implying rapid degradation of POC in the hypolimnion. The ¹³C of DIC added to the deep water (-22.) was too heavy to have been derived from oxidation of particulate organic carbon alone. About 20% of the added DIC must have diffused from hypolimnetic sediments where relatively heavy CO₂ (-7) was produced by a combination of POC oxidation and as a by-product of methanogenesis.     

N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

Use of a Novel Type of Rotating Disc Electrode and a Flow Cell with Laminar Flow Pattern for the Electrochemical Detection of Biogenic Monoamines and Their Metabolites After Sephadex Gel Chromatographic Purification and High Performance Liquid Chromatographic Isolation from Rat Brain

Abstract: A novel type of rotating disc electrode and a flow cell with laminar flow pattern were developed and applied to the electrochemical detection of dopamine, 3,4-dihy-droxyphenylacetic acid, homovanillic acid, 3-methoxytyra-mine (3-MT), noradrenaline, 3-methoxy-4-hydroxyphenyl-ethyleneglycol (MOPEG), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid after HPLC of these compounds. The active surface of the rotating disc working electrode was made from solid paraffin (40%; wt/wt) and graphite powder (60%; wt/wt). The sensitivity of the detector was proportional to the square root of the angular velocity and was practically independent of the flow rate of the mobile phase. The surface of the working electrode was very large (radius = 12 mm), and so the percentage of oxidation was 24–67%; (flow rate = 1.0 ml/min), depending on the compound. Electrical noise between 20 and 40 pA and background current of 20–60 nA were observed. In practice, the sensitivity for the detection of the compounds examined here was 8–16 nA/ng, and so a detection limit of 5 pg/injection could be achieved, when the detector was combined with reversed-phase HPLC. Supernatants obtained from the extracts of the tissue samples (nine brain parts of rat brain were studied) were purified by using Sephadex G-10 gel chromatography. Before this procedure, the proteins of the tissue extracts were precipitated by 0.2 M HC1O₄, and the excess of HC1O₄ was precipitated by KOH/HCOOH buffer. Simultaneously, the pH of the extracts was set to 2.4 by the above buffer. Adjustment of the pH was necessary so that elution of 5-HT from the Sephadex G-10 columns in the same fraction with 3-MT was avoided. If these compounds were in the same solution, their peaks would overlap on HPLC. MOPEG sulfate was purified by diethylaminoethyl-Sephadex A-25 (anion exchange resin) from the first fraction collected from the Sephadex G-10 columns. The contents of the compounds under investigation in nine brain parts agreed with those found by other investigators.     

用薄层色谱分离提纯单半乳糖和双半乳糖双甘油酯

本文选择两种溶剂体系,用两次单向薄层层析,从小麦抗寒与不抗寒品系天然橡胶胶乳分离提纯出6种单半乳糖和双半乳糖双甘油酯。并比较了它们的疏水侧链的脂肪酸组成。小麦糖脂疏水侧链脂肪酸的不饱和指数远大于天然胶乳糖脂。抗寒品系胶乳糖脂疏水侧链脂肪酸不饱和指数大于不抗寒品系。双半乳糖双甘油酯疏水侧链脂肪酸不饱和指数均大于其单半乳糖糖脂。