Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti

A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.     

Mg2+-paracrystal formation of tropomyosin as a condensation phenomenon. Effects of pH, salt, temperature, and troponin binding.

Tropomyosin (Tm) paracrystal formation induced by Mg2+ was studied by monitoring increases in light scattering. Paracrystals formed above a critical Tm concentration with lag phases in the time courses at pH 7.5 and 6.0, indicating that condensation polymerization processes are involved. The kinetic data at pH 7.5 reasonably fit a model in which nucleation and elongation are taken into account. The rate and extent of light scattering increased at low [Mg2+] and decreased at high [Mg2+] with a maximum at [Mg2+] = 15 mM, indicating different effects of Mg2+ in the two [Mg2+] ranges. The paracrystals were destabilized by increasing the salt concentration and decreasing the temperature. Mg2+ produces paracrystals at pH 6.0 and pH 7.5 by different kinetic mechanisms. Different Tm intermolecular interactions at the two pH values were indicated by studies of the excimer fluorescence of pyrene-labeled Tm and by effects of salt and temperature on the kinetics. At pH 6.0 Tm more readily formed paracrystals with decreased electrostatic effects. Effects of troponin on Mg2+-paracrystal formation of Tm at the two pH values correlated with the known differences in paracrystal structure when troponin is bound to Tm.     

Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

Requirement of protein co-factor for the DNA-binding function of the human ski proto-oncogene product.

We identified the human c-ski gene product (c-Ski) as a protein with the apparent molecular weight of 100,000, p100c-ski, by using a c-Ski-specific polyclonal antibody. p100c-ski was a nuclear protein and p100c-ski in nuclear extracts of Molt4 cells bound to calf thymus DNA cellulose, but the bacterially synthesized c-Ski did not, suggesting that Ski was associated with another protein(s) and that the Ski complex had DNA-binding activity. This hypothesis was supported by the finding that the bacterially synthesized Ski bounds to DNA cellulose after being mixed with a nuclear extract of Molt4 cells. By use of a series of deletion mutants of Ski synthesized in an in vitro translation system, two portions in Ski were found to be necessary for the DNA binding of the Ski complex: the N-proximal portion containing a cystein/histidine-rich domain and the C-terminal portion including a region rich in basic amino acids.     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.     

Increased renal TXA2 synthesis in diabetes mellitus: simultaneous determination of urinary TXB2 and 2,3-dinor-TXB2

The present study was designed to determine urinary excretion of kallikrein(KAL)-kinin as well as prostaglandin (PG) E2, TXB2 and 2,3-dinor-TXB2, a major urinary metabolite of TXA2 synthesized in platelets, by specific RIAs in patients with diabetes mellitus (DM). KAL or kinin excretion in 26 type II DM did not differ from control values obtained in 18 age-matched healthy subjects (C), although DM with HbA1 greater than 11% excreted less KAL. Urinary PGE2 excretion (7.6 +/- 2.8 ng/mg creatinine, mean +/- SE) was significantly lower in DM compared to C (17.5 +/- 3.9, p less than 0.05), while DM excreted more TXB2 (0.57 +/- 0.09, p less than 0.01) and 2,3-dinor-TXB2 (0.56 +/- 0.12, N.S.) than C (0.19 +/- 0.02 or 0.33 +/- 0.01). DM with or without mild proteinuria demonstrated lower PGE2, but higher TXB2 and 2,3-dinor-TXB2 excretion. A positive correlation of TXB2/2,3-dinor-TXB2 with proteinuria was observed in this group. However, in DM with massive proteinuria over 500 micrograms/mg creatinine, TXB2 and 2,3-dinor-TXB2 excretion decreased to levels almost identical to C. As a whole, a ratio of TXB2 to PGE2 or 2,3-dinor-TXB2 in DM was significantly higher than in C. The results suggest that a relative preponderance of TXB2 to 2,3-dinor-TXB2 may indicate an augmented renal, in addition to platelet, TXA2 synthesis. An excessive vasoconstrictive and proaggregatory TXA2 renal synthesis, concomitant with a decrease in vasodilatory and antiaggregatory PGE2, may have profound effects on renal functions such as protein excretion in DM.