Biosynthesis and glycosylation of the carcinoembryonic antigen.

Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.     

The effect of alkaline pH on the cell growth of six different mammalian cells in tissue culture

The effect of high alkaline pH on the reinitiation of cell growth was studied in six different mammalian cells. We failed to confirm the observation of Zetterberg & Engström, Proc natl acad sci US 78 (1981) 4334 [17] and Exp cell res 144 (1983) 199 [18]. Treatment of quiescent cells at pH 9.5 did not stimulate cell growth when measured by total protein/flask or increase in cell number.     

Characterization of thermotropic structural transitions of the erythrocyte membrane: a biochemical and electron-paramagnetic resonance approach

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.     

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.     

Protein-ligand interaction. A calorimetric study of the interaction of oligosaccharides and hen ovalbumin glycopeptides with concanavalin A

A calorimetric study is reported concerning the interaction between concanavalin A (Con A) and some oligosaccharides and glycopeptides hydrolyzed from hen ovalbumin. The measurements were carried out in acetate buffer, pH 4.5, where, by far, the prevailing form of the protein is the dimeric one [Kalb, A.J., & Lustig, A. (1968) Biochim. Biophys. Acta 168, 366; Dani, M., Manca, F., & Rialdi, G. (1981) Biochim. Biophys. Acta 667, 108]. The calorimetric technique allows the direct determination of the binding enthalpy delta H, degrees B, the evaluation of the apparent association constant K'B, and then the evaluation of the apparent free energy and entropy, delta G degrees' B and delta S degrees' B. Three groups of data have been collected in the present study. The first one concerns the interaction between concanavalin A and some mono- and disaccharides [methyl alpha-glucopyranoside (alpha MGlup), methyl alpha-mannopyranoside (alpha MManp), D-maltose, D-trehalose, and D-cellobiose]. The analysis of the data indicates that in these cases there are small favorable entropic and enthalpic contributions to the affinity. The stoichiometry of the reaction is 2 mol of ligand/mol of Con A dimer, the sites resulting being equivalent and noninteracting. Melezitose, the only trisaccharide studied, shows a different behavior: its affinity for Con A is higher as compared to the other oligosaccharides containing alpha-glucosyl residues and closer to that of methyl alpha-mannopyranoside. However, the stoichiometry is different, namely, 1 mol of ligand/dimer of Con A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the first adult de novo case of 46,X,der(Y)t(X;Y)(p22.3;q11.2)

Herein, we describe a case of an infertile man detected in postnatal diagnosis with FISH characterization and array-CGH used for genome-wide screening which allowed the identification of a complex rearrangement involving sex chromosomes, apparently without severe phenotypic consequences. The deletion detected in our patient has been compared with previously reported cases leading us to propose a hypothetical diagnostic algorithm that would be useful in similar clinical situations, with imperative multi disciplinary approach integrated with genetic counseling. Our patient, uniquely of reproductive age, is one of six reported cases of duplication of Xp22.3 (~ 8.4 Mb) segment and contemporary deletion of Yq (~ 42.9 Mb) with final karyotype as follows:

46,X,der(Y),t(X;Y)(Ypter → Yq11.221::Xp22.33 → Xpter).ish der(Y) (Yptel+,Ycen+,RP11-529I21+,RP11-506M9-Yqtel −,Xptel +). arrXp22.33p22.31(702–8,395,963, 8,408,289x1), Yq11.221q12 (14,569,317x1, 14,587,321–57,440,839x0)     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Cadmium uptake,localization and stress-induced morphogenic response in the fern Pteris vittata

Cadmium uptake, tissue localization and structural changes induced at cellular level are essential to understand Cd tolerance in plants. In this study we have exposed plants of Pteris vittata to different concentrations of CdCl₂ (0, 30, 60, 100 μM) to evaluate the tolerance of the fern to cadmium. Cadmium content determination and its histochemical localization showed that P. vittata not only takes up, but also transports and accumulates cadmium in the aboveground tissues, delocalizing it mainly in the less bioactive tissues of the frond, the trichomes and the scales. Cadmium tolerance in P. vittata was strictly related to morphogenic response induced by the metal itself in the root system. Adaptive response regarded changes of the root apex size, the developmental pattern of root hairs, the differentiation of xylem elements and endodermal suberin lamellae. All the considered parameters suggest that, in our experimental conditions, 60 μM of Cd may represent the highest concentration that P. vittata can tolerate; indeed this Cd level even improves the absorbance features of the root and allows good transport and accumulation of the metal in the fronds. The results of this study can provide useful information for phytoremediation strategies of soils contaminated by Cd, exploiting the established ability of P. vittata to transport, delocalize in the aboveground biomass and accumulate polluting metals.     

Identification of a novel glycopeptide species that correlates with differentiation of F9 cells

The high-molecular-weight fucosyl glycopeptides of differentiated F9 cells have been analyzed. We found that these high-molecular-weight surface structures contain two components with different molecular weights, the largest of which, peak I, has never before been reported. The material eluting in this peak seems to contain only acidic species. Removal of sialic acid from both the peak I and the peak II species does not eliminate the differences in molecular weight, indicating that the two species have more profound structural differences than can be accounted for by sialic acid. Since peak I glycopeptides were found both in differentiated F9 cells and in two parietal endoderm cell lines, we suggest that its presence is related to parietal endoderm differentiation.