Fast atom bombardment tandem mass spectrometric analysis of N-carbamoylamino acids.

Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase and T cell receptor alpha subunit are differentially degraded in the endoplasmic reticulum.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is located in the endoplasmic reticulum (ER) and responds to rapid degradation which is regulated by mevalonate or sterols. T cell antigen receptor alpha chain (TCR alpha) is also known to be rapidly degraded within the ER. In both cases, the membrane domains of the proteins have a crucial role in their rapid degradation. In order to investigate protein degradation in the ER, we compared the degradation of HMG-CoA reductase and TCR alpha in the same Chinese hamster ovary cells. Among the protease inhibitors tested, N-acetyl-leucyl-leucyl-methioninal blocks the degradation of HMG-CoA reductase and also inhibits the degradation of TCR alpha. On the other hand, N-tosyl-L-phenylalanine chloromethyl ketone and N-carbobenzoxy-L-phenylalanine chloromethyl ketone inhibit the degradation of TCR alpha but have no effect on the degradation of HMG-CoA reductase. Diamide, a thiol-oxidizing agent, blocks the degradation of both HMG-CoA reductase and TCR alpha. Perturbation of cellular Ca2+ attenuates the rapid degradation of HMG-CoA reductase but does not affect the degradation of TCR alpha. Furthermore, thapsigargin, a selective ER Ca(2+)-ATPase inhibitor, and Co2+, a potent Ca2+ antagonist, increase the half-life of HMG-CoA reductase but not that of TCR alpha. Energy inhibitors diminish the rapid degradation of HMG-CoA reductase but not that of TCR alpha. These results suggest that although HMG-CoA reductase and TCR alpha appear to be degraded in the same subcellular compartment, the mechanisms responsible for degradation differ.     

Induction of IL-2 receptor expression and proliferation of T cell clones by a novel cytokine(s).

We found a unique T cell IL-2 receptor (IL-2R)3-inducing activity in the supernatant (SN) of the TH1 clone stimulated with antigen on spleen cells as antigen-presenting cells (APC). We have tentatively named this activity the IL-2R-inducing factor (IL-2RIF) and have characterized the activity. The SN induced IL-2R and proliferation of TH1 clones stimulated with B cell APC, which could not induce IL-2R in the absence of the SN. Other known cytokines were examined for a IL-2RIF activity; however, none of cytokines examined exerted a similar activity. Moreover, the neutralizing antibodies against the known cytokines tested did not block the IL-2RIF activity in the SN. When TH1 clones were stimulated with immobilized anti-CD3 or with fixed B cell APC in the presence of partially purified IL-2RIF, these clones expressed IL-2R and showed IL-2-dependent proliferation, whereas they induced neither IL-2R expression nor proliferation in the absence of IL-2RIF activity. These observations suggest that IL-2RIF activity is mediated by a novel cytokine(s) and the cytokine plays an important role as a second signal in the activation of the TH1 clone.     

Peptide transport by the multidrug resistance pump.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.     

Calcium binding to phospholipid: structural study of calcium glycerophosphate.

To consider possible interaction of the phospholipid membrane with calcium ions, crystal structures of calcium dl-alpha- and beta-glycerophosphates (alpha- and beta-CaGs, respectively) were investigated by X-ray diffraction methods. After many attempts, relatively large single crystals of beta-CaG were prepared from the aqueous solution containing HCl, while crystals of CaHPO4.2H2O were obtained from alpha-CaG solution under the same crystallization conditions. The crystal structure of beta-CaG is orthorhombic with space group Pna2(1) and cell dimensions of a = 8.251(1), b = 13.038(3), c = 25.483 (10) A, V = 2741.5 (13) A3 and Z = 16 [four molecules (A to D) in an asymmetric unit]. Molecules of A to D took, as a whole, similar extended conformations, although A and B were different from C and D in the orientation about a glycerol C-C bond. Four independent beta-glycerophosphates commonly act as two types of bidentate ligands, where one is the coordination to the calcium ion by the glycerol O(1) and phosphate O(22) atoms, and the other by the phosphate O(22) and O(23) atoms, thus forming the calcium coordination of a distorted square plane, respectively. Each of four independent calcium ions forms the same coordination geometry of a distorted pentagonal bipyramid. Infinite double layers consisting of alternate A/B molecules and of alternative C/D ones and sandwiching calcium ions were arranged face-to-face along the b-direction and were piled up in the a-direction, thus forming the stacked bilayer unit with the thickness of d002 = 12.75 A. The elaborate networks of calcium coordinations and hydrogen bondings were formed among the layers and stabilized the crystal structure. Based on the structural parameters of the present beta-CaG crystal, a possible interaction model of phospholipid with calcium ions was proposed.     

The hypnogenic effects of delta sleep-inducing peptide (DSIP) analogs: a comparative study in rabbits and rats]

Hypnogenic effects of 3 DSIP analogs with a higher stability against aminopeptidase activity have been studied in rabbits and rats using intraventricular administration (injections and infusions). An analog (D-Ala-2) DSIP augmented slow wave and paradoxical sleep within the 5th, 8th and 11th hours of the recording period. An analog (D-Val-2) DSIP made the same within the 8th and 10th hours, and hexapeptide (D-Ala-2) DSIP (1-6) increased sleep during the 1st, 3rd, and 5th hours. Both nonapeptides augmented sleep in rabbits as well as in rats, though hexapeptide produced this effect in rabbits only, that might be related to some difference in distribution and colocalization of endogenous DSIP-like peptide in the pituitary of two rodent species. It may be suggested that hypnogenic activity of DSIP analogs is determined by the structure of administrated molecule, being mediated by such hormones as GRF and CLIP.     

Biosynthesis of poly(3-hydroxyalkanoate) from amino acids

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.     

Production of S-lactoylglutathione by organic-solvent-extracted glyoxalase I from Hansenula mrakii

Summary Glyoxalase I was extracted from Hansenula mrakii IFO 0895 by incubating the cells with buffer solution containing 50% acetone (enzyme activity 35 units/g cells) or 50% ethyl acetate (enzyme activity 28 units/g cells) at 30°C for 10 h. Glyoxalase II was also extracted from the cells, although the activity of the enzyme was lost during incubation with organic solvents, especially at higher temperature (30°C). By using the organic-solvent-extracted fraction of H. mrakii, enzymatic production of S-lactoylglutathione was studied, and approximately 82 mmol/l (30 g/l) of S-lactoylglutathione was produced from 120 mmol/l glutathione. Offprint requests to: A. Kimura     

Identification of free glycan chain liberated by de-N-glycosylation of the cortical alveolar glycopolyprotein (hyosophorin) during early embryogenesis of the Medaka fish, Oryzias latipes

In Medaka embryos (at the stages of blastulation to organogenesis), we found the presence of free glycan of which structure is identical with the multiantennary N-linked sugar chain of L-hyosophorin molecules which were originally present in the cortical alveoli of the unfertilized eggs in their precursor high molecular form. The free glycan-enriched fraction was separated from L-hyosophorin by chromatography on DEAE-Sephadex A-25 and Sephadex G-50 after removal of the sialic acid residues with exo-sialidase. Composition analysis, 400-MHz 1H NMR spectroscopy, and pyridylamination-hydrazinolysis-nitrous acid deamination of the free glycan showed the presence of di-N-acetylchitobiosyl structure at the reducing end, suggesting that the free glycan chain was derived from L-hyosophorin by the action of a specific peptide:N-glycosidase (PNGase). When we combine the previous finding of the hyosophorin-derived unique pentaantennary free glycan chain in the flounder embryos [A. Seko et al. (1989) J. Biol. Chem. 264, 15922-15929], it is anticipated that PNGase-catalyzed de-N-glycosylation of L-hyosophorin would be required at a certain stage of embryogenesis for L-hyosophorin to play a yet undefined functional role during early development.     

Somatostatin inhibits the nicotinic receptor-activated inward current in guinea pig chromaffin cells.

At a holding potential of -60 mV, bath application of somatostatin produced no current response itself, but inhibited the nicotinic inward current, a concentration of about 0.01 microM being required for a half-maximal effect. This inhibition was affected neither by pretreatment with somatostatin nor by injection of guanosine-5'-0-(3-thiotriphosphate) through a patch pipette. When somatostatin (0.03-0.1 microM) produced its maximum inhibition, the proportion of plateau to peak nicotinic current was greatly reduced (to 5.3 +/- 5.7% from the control value of 34.9 +/- 4.9%, n = 5) with a facilitation of the time course of desensitization. After washout of somatostatin, the nicotinic current was restored to control level in a time-dependent, rather than an activation-dependent manner. The results suggest that somatostatin inhibits the function of the nicotinic receptor by facilitation of its desensitization or by an open-channel block.