Purification to apparent homogeneity of the somatostatin receptor of the human cell line HGT-1 of gastric origin

This communication reports the isolation and the purification of the gastric somatostatin receptor from the human cell line HGT-1. The receptor has been extracted from the cell membrane by Triton X 100, and a monoclonal antibody to this was prepared. A series of affinity chromatographies (Sepharose-antibody and Sepharose-somatostatin-14) and a final purification by steric exclusion on high performance liquid chromatography columns (HPLC) allowed us to obtain a fraction enriched 20,000 fold in 125I-Tyrll-somatostatin-14 specific binding (apparent dissociation constant: 7.6 x 10(-8) M). This fraction corresponded to a molecular mass of about 90 kDa (in presence of detergent) and to a maximal binding capacity of more than 10,000 pmol/protein. It therefore has a theoretical homogeneity close to 100%.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.     

Sensory receptors of the ovipositor ofTrichogramma maidis [Hym.:Trichogrammatidae]

Sensory receptors of the ovipositor ofTrichogramma maidis are described. Sense organs are found on the 2^nd valvifers (1 type), on the tip of the 3^rd valvulae (2 types) and on the 1^st valvulae (4 types). The nature and possible functions of these sensilla are discussed.      

A cellular model for drug interactions on hematopoiesis: The use of human umbilical cord blood progenitors as a model for the study of drug-related myelosuppression of normal hematopoiesis

A cellular model of hematopoiesis which would be more convenient than bone marrow (BM) progenitors and directly relevant to human pathology is needed in order to investigate xenobiotic toxicity. Human umbilical cord blood (HCB), previously shown to be able to repopulate BM, provides a powerful in vitro model of normal human hematopoiesis. In order to validate the use of normal HCB progenitors as targets for dose-related myelosuppression, we used clonogenic assays and expansion in a liquid culture of progenitor-enriched cell suspensions from HCB. A series of 8 reference molecules, doxorubicin, cytosine-arabinoside, 5-fluorouracil, 3-azido-3-deoxythymidine, acetylsalicyclic acid, sodium valproate and two cephalosporin antibiotics, were tested. In vitro 50% inhibition concentrations (IC₅₀) were compared to those observed or reported with BM progenitors, and to the values of plasma concentrations from treated patients. HCB progenitors as in vitro targets for cytotoxic molecules were easy to access and handle, and their use was sensitive, specific and reproducible. They gave results similar to BM progenitors and allowed a qualitative approach to cellular metabolism and toxicity using morphological, flow cytometric and chromatographic methods.Abbreviations ARA-CC cytosine arabinoside - AS acetylsalicylic acid - AZTT 3-azido-3-deoxythymidine - BFUU burst-forming units - BM bone marrow - CFU colony-forming units - DOXX doxorubicin - FU 5-fluorouracil - glyAA glyAcophorin A - HCB human umbilical cord blood - IU international units - PCMEM human placenta-conditioned medium - VA sodium valproate     

Conformational properties of Eu(III) complexes of 3,3′; 5,3″-polymethylene bridged derivatives of 2,2′; 6,2″-terpyridine

The complex [Eu(tpy)₃](ClO₄)₃ where TPY=2,2′; 6,2″-terpyridine, has been prepared and reexamined. The complex appears to be stable in acetonitrile solution with respect to decomplexation of the ligands but the addition of water does cause partial replacement of tpy. Analogous complexes have been prepared with 3,3′; 5,3″-polymethylene bridged derivatives of tpy having two or three carbons in the bridge. The bridging enforces a cisoid geometry of the ligand and prohibits its replacement by added water. An X-ray determination was carried out for [Eu(3b)₃](ClO₄)₃, where 3b=3,3′; 5,3″-dimethylene tpy, which crystallizes in the monoclinic space group P2₁/c with a=11.908(4), b=15.768(5), c=29.513(9) Å, β=93.60(2)°, μ=13.5 cm⁻¹ and Z=4. The complex forms a tricapped trigonal prism with each of the ligands adopting the same dl conformation. Variable temperature NMR analysis of the bridged ligand complexes indicates that conformational inversion of the bound ligand is not a concerted process and barriers for inversion of individual methylene units can be estimated from coalescence of the signals from the geminal methylene protons. The luminescence properties of the bridged tpy complexes are similar to the parent unbridged system.