Limitations of Chromogranin A in clinical practice

Context: Usefulness of circulating Chromogranin A (CgA) for the diagnosis of neuroendocrine tumors (NEN) is controversial. The aim of the present study was to assess the actual role of this marker as diagnostic tool. Methods: Serum blood samples were obtained from 42 subjects affected with NEN, 120 subjects affected with non-endocrine neoplasias (non-NEN) and 100 non-neoplastic subjects affected with benign nodular goitre (NNG). Determination of CgA was performed by means of immunoradiometric assay. Results: The CgA levels among NEN-patients were not significantly different from NNG and non-NEN subjects. The Receiver operating characteristic (ROC) curves analysis failed to identify a feasible cut-off value for the differential diagnosis between NEN and the other conditions. Conclusion: Serum CgA is not helpful for the first-line diagnosis of NEN.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Essential role of MESP1-RING1A complex in cardiac differentiation

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In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Electrophysiological evidence for the autoregulation of β-cell secretion by insulin

Electrophysiological studies of cultured rat pancreatic β-cells using intracellular microelectrodes show that exogenous insulin over the range of 0.1–10.0 μg/ml inhibits the electrical activity due to 27.8 mM glucose in a dose-related manner. This inhibitory effect is manifested by a mean increase of the membrane potential from about ?20 to ?30 mV and inhibition of the manner of cells impaled showing spike activity from 60 to less than 10%. The inhibitory influence of insulin is rapid occuring within 5 min for the highest level used. The results provide evidence for a negative feedback role of insulin in regulating its own release.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Changing behavior of surface immunoglobulin on mouse B lymphocytes during immune development: I. LPS-induced changes in the lateral mobility of B lymphocyte surface immunoglobulins on two populations which express different surface immunoglobulin phenotypes

The redistribution of surface membrane immunoglobulin molecules (sIg) was studied in two functionally distinct populations of mouse splenic B lymphocytes, namely, those bearing membrane IgM(IgG^?) and those bearing IgG. Brief exposure to mitogenic doses of bacterial lipopolysaccharide (LPS) produced direct but differential effects on the subsequent ability of specific antibodies to induce this redistribution on each cell type. Studied as a function of temperature, antibody-induced redistribution of sIgM on cells previously exposed to LPS was observed to occur at temperatures lower than the temperatures required for similar sIgM redistribution on lymphocytes not exposed to LPS. In contrast, mitogen-treated sIgG⁺ cells demonstrated an opposite and long-lasting effect (at least 40 hr), requiring higher temperatures to allow sIgG movement comparable to that seen on untreated sIgG-bearing lymphocytes. Thus, we conclude that LPS interacts with both IgM⁺(IgG^?) and IgG⁺ lymphocytes, but that such interactions produced different membrane effects on each B-cell subset. This membrane change can therefore be useful as a quasi-functional differentiation marker. Furthermore, differences in sensitivity to cellular activation by LPS seen between sIgM-bearing (sIgG^?) and sIgG-bearing B cells may be a reflection of such direct, although different, membrane effects.     

Analysis of the effects of cesium ions on potassium channel currents in biological membranes

Cesium ions block potassium channels in biological membranes in a voltage dependent manner. For example, external cesium blocks inward current with little or no effect on outward current. Consequently, it produces a characteristic N-shaped current-voltage relationship. We have modeled this result by single file diffusion of ions in a narrow channel spanning the membrane with a special blocking site in the channel for cesium ions. The model enables us to make detailed comparisons of the effects of cesium on potassium channels in different types of biological membranes.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.