Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Assignment of the human myeloperoxidase gene (MPO) to bands q21.3----q23 of chromosome 17

Using a human myeloperoxidase cDNA, we have mapped the human myeloperoxidase gene to chromosome 17 at q21.3----q23 by in situ hybridization to metaphase chromosomes from human lymphocyte preparations.     

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.     

ESR study on synthetic glyceroglycolipid liposomal membranes

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmitoyl[beta-cellobiosyl-(1'---3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37 degrees C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-6-DPPA in the Cel-DAG liposomes at 37 degrees C showed broad resonance lines with the central peak being the highest, while that at 60 degrees gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37 degrees C but not above Tc. These results suggest that below Tc the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.     

Developmental expression and intracellular location of P400 protein characteristic of Purkinje cells in the mouse cerebellum

The developmental expression and intracellular localization of a cerebellum-characteristic 250-kDa glycoprotein, P400 protein, were studied by immunohistochemical and immunoblot methods using a monoclonal antibody against P400 protein. In the cerebellum of normal mouse, the expression of P400 protein increased from Postnatal Day 3 to Day 21. This enhancement of P400 protein expression occurred only in the Purkinje cells and proceeded with the growth of their dendritic arborization. Electron microscopic analysis indicated that P400 protein is present at the plasma membrane, the endoplasmic reticulum, and the postsynaptic densities of Purkinje cells. Immunohistochemistry of the cerebella of neurological mutant mice indicated that the Purkinje cells of reeler, weaver, and pcd mutant mice retain the ability to produce a large amount of P400 protein. However, the Purkinje cells of staggerer mutant mouse proved to be incapable of enhanced P400 protein expression. These results indicate that P400 protein is a Purkinje cell-characteristic plasma membrane-associated glycoprotein, which is also present at the postsynaptic density and endoplasmic reticulum and that the expression of P400 protein in Purkinje cells is closely associated with the growth of their dendritic arborization.     

Effects of pancreastatin on insulin and pancreatic polypeptide secretion in the dog

Porcine pancreastatin (1.19 nmol) was administered into the peripheral vein (i.v.) or the third cerebral ventricle (i.t.v.) of dogs and its effect on the secretion of insulin and pancreatic polypeptide (PP) studied. Neither means of administration had any effect on basal and glucose-induced insulin or PP secretion. However, i.v. pancreastatin did inhibit the i.v. CCK-8-induced insulin but not PP release. Pancreastatin may thus play a role in the regulation of insulin secretion in the canine pancreas.     

Degradation of 125I-glucagon, -pancreatic polypeptide and -insulin by acid saline extract of rat submaxillary gland and their protection by proteinase inhibitors

The acid saline extract (ASE) of rat submaxillary gland exerts a powerful degrading effect on 125I-glucagon. In order to study the degradation of other 125I-peptides by ASE and the effects of their inhibitors, 125I-pancreatic polypeptide (PP) and 125I-insulin were used together with 125I-glucagon. The degradation studies were done by the trichloroacetic acid (TCA) method or gel filtration. Besides 125I-glucagon, 125I-PP was found to be destroyed by ASE in the ordinary immunoassay system using the TCA method, but 125I-insulin was intact in the presence of ASE. Leupeptin, and to a lesser extent p-chloromercuriphenyl-sulfonic acid (PCMS) and N-ethylmaleimide, inhibited the destruction of 125I-glucagon or -PP under the TCA method. PCMS was especially protective at high concentrations, for example 16 mM. These findings were confirmed by gel filtration of the assay mixture. In the presence of leupeptin (0.4 mM) and PCMS (16 mM), no shift in the peak of labelled glucagon or PP occurred. Thus ASE degrades not only 125I-glucagon but -PP, and thiol proteinase inhibitors have a strong inhibitory action on them.     

Radioiodine (131I) therapy of Graves' disease: use of the new high resolutional ultrasonic scanner for the determination of the accurate weight of the thyroid gland

In this series, eighteen patients with Graves' disease were treated with 8000 rads (80 Gy) of radioiodine (131I), using the new high resolutional ultrasonic scanner for the determination of the accurate weight of the thyroid gland. The mean dose of radioiodine administered orally was 4.6 +/- 3.0 mCi (170.2 +/- 110.0 MBq) and 133.7 +/- 44.6 microCi/g (4.95 +/- 1.65 MBq). At one year after treatment, twelve of eighteen patients (66.7%) became euthyroid, five (27.8%) remained hyperthyroid and one (5.6%) became hypothyroid. Analysis of various factors which may be related to the effect of radioiodine therapy revealed that the weight of the thyroid gland in the hyperthyroid and euthyroid groups was significantly different (61.7 +/- 33.5 g vs. 25.1 +/- 9.1 g, p less than 0.05). Furthermore, all patients with larger glands (more than sixty grams) remained hyperthyroid, while the incidence of euthyroidism was as high as 80% in patients with smaller glands (less than forty grams). Although the number of patients studied was small, these results indicate that a larger thyroid gland requires a larger radioiodine dose per gram of tissue than a smaller gland, suggesting that the therapeutic radiation dose should be graded according to the gland size even when the gland size is accurately estimated by ultrasound. Further study is required to determine the appropriate radiation dose graded according to the gland size.     

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.     

Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.