Mario F. Tecce

PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus.

We have used differential display to identify genes whose expression is altered in type 2 diabetes thus contributing to its pathogenesis. One mRNA is overexpressed in fibroblasts from type 2 diabetics compared with non‐diabetic individuals, as well as in skeletal muscle and adipose tissues, two major sites of insulin resistance in type 2 diabetes. The levels of the protein encoded by this mRNA are also elevated in type 2 diabetic tissues; thus, we named it PED for phosphoprotein enriched in diabetes. PED cloning shows that it encodes a 15 kDa phosphoprotein identical to the protein kinase C (PKC) substrate PEA‐15. The PED gene maps on human chromosome 1q21–22. Transfection of PED/PEA‐15 in differentiating L6 skeletal muscle cells increases the content of Glut1 transporters on the plasma membrane and inhibits insulin‐stimulated glucose transport and cell‐surface recruitment of Glut4, the major insulin‐sensitive glucose transporter. These effects of PED overexpression are reversed by blocking PKC activity. Overexpression of the PED/PEA‐15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Antioxidant effect of hydroxytyrosol (DPE) and Mn2+ in liver of cadmium-intoxicated rats.

Liver TBARS formation in cadmium-intoxicated rats was completely reduced by administering a low amount of MnCl(2) (2 mg/kg b.w.) 1 h before intoxication. A similar antioxidant effect was first shown by hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, given twice to rats (9 mg/kg b.w.) after cadmium administration. The antioxidant properties shown in vivo by both Mn(2+) and DPE were also active in vitro when rat liver microsomes were subjected to lipid peroxidation by cadmium or other prooxidant systems. The increase in liver glutathione concentrations occurring in cadmium-intoxicated rats, was also found, for the first time, 24 h after MnCl(2) administration. Unlike cadmium intoxication, which caused a higher formation of both glutathione and TBARS, Mn(2+) induced glutathione synthesis without any TBARS formation. The same situation was also observed when cadmium plus Mn(2+) or cadmium plus DPE was given to rats. Our data show that: (a). both DPE and low Mn(2+) concentrations may have an antioxidant effect in the livers of cadmium-intoxicated rats and (b). Mn(2+), like cadmium, induces liver glutathione synthesis and this effect is probably independent of TBARS formation.

A nonincinerative rate-sensing method for the determination of iodine in iodoproteins

Abstract The method described here for iodine determination in iodoproteins deals both with a nonincinerative treatment of samples and with the application of a kinetic method of analysis to the iodine-catalyzed ceric-arsenite redox reaction. This method represents an improvement of existing methods, with new significant modifications, which applies especially to thyroid iodoproteins in the range of 10–1000 pmol of iodine. The procedure proposed is essentially composed of two steps: (1) treatment of samples with bromine in an acidic solution; (2) colorimetric estimation of the iodine produced by measuring the rate of decrease of the cerium(IV) color. The change in absorbance at 410 nm, due to the reduction reaction, is monitored photometrically. The reaction rate, which is proportional to the concentration of iodine, is derived from the slope of the absorbance change plotted versus time. The method allows: (i) a significant time saving, since the procedure requires less than 30 min; (ii) the use of optical plastic cuvettes as reaction cells, so that any transfer of sample is avoided; (iii) the use of common laboratory equipment, no special ashing or analytical apparatus being necessary. Moreover, the kinetic approach ensures precision and accuracy of the measurements. This procedure is proposed as a simple process for the sensitive determination of the iodine in iodoproteins without separation or the need for alkaline or acid digestion. The procedure is also suitable for iodine salts and iodoamino acids.

High-yield and high-degree purification of human α-fetoprotein produced by adaptation of the human hepatoma cell line Hep G2 in a serum-free medium

The human hepatoma cell line Hep G2 secretes both albumin and alpha-fetoprotein when grown in the presence of serum. The present report describes how adaptation to growth in serum-free medium results in a progressive switch in the expression of the two proteins; i.e., alpha-fetoprotein becomes the main protein secreted while albumin production is greatly reduced. The culture supernatant obtained, being very enriched in the protein, allows the development of a purification procedure by preparative electrophoresis. By this procedure it is possible to easily obtain large amounts of alpha-fetoprotein from a constant and unlimited source. The availability of these protein preparations should improve the reproducibility and the quality of standardization in clinical immunoassays for alpha-fetoprotein and should permit a more accurate study of the structure and biological functions of the protein.

A four- to sixfold enhancement in sensitivity for detecting trace proteins in dye or silver stained polyacrylamide gels.

A very simple procedure which enhances sensitivity in visualizing proteins stained by all standard procedures in polyacrylamide gels is described. The process, based on the use of concentrated polyethylene glycol 6000 solutions, produces a conspicuous, reversible, and uniform size reduction of gels. The entity of the reduction depends principally on the polyethylene glycol 6000 concentration and on the acrylamide content of gels. Using an appropriate polyethylene glycol 6000 concentration, it is possible to regulate the final size and then the sensitivity as needed. Depending upon the above conditions it is possible to increase the sensitivity by a factor of 4-6 or more. Reduction of the gel volume by such a factor may eliminate the need for the cumbersome and frequently impossible preconcentration of samples. Besides the Coomassie blue stained gels, silver stained gels may also be treated in a similar manner with a consequent further enhancement in sensitivity. In addition, gels containing radiolabeled materials can be autoradiographed in times proportionally shorter, without significant sacrifice of resolution.

Probing the interaction of thyroglobulin with metal ions by terbium(III) luminescence spectroscopy.

The binding of Ca2+ ions to bovine and human thyroglobulin (Tg) was demonstrated qualitatively by 45Ca overlay on polyvinylidene difluoride (PVDF) membranes. A quantitative analysis of the interaction of metal ions with bovine Tg was conducted by fluorimetric titration of the protein with Tb3+ ions. These have been used with several proteins as isomorphous replacement probes for Ca2+ ions, as protein-bound Tb3+ ions fluoresce, upon irradiation in the UV region, because of energy transfer from tyrosyl and/or tryptophanyl residues. The fluorescence emission spectrum of Tg excited at 280 nm showed, upon addition of Tb3+ ions, a peak at 546 nm and a marked decrease at 335 nm, indicating an efficient Förster energy transfer between bound Tb3+ ions and closely located Tg intrinsic chromophores. Titration of Tg with Tb3+ ions, carried out by monitoring the emitted fluorescence at 546 nm, indicated the presence of 13.15 metal binding sites per Tg molecule.

Molecular organization of 19 S calf thyroglobulin

Controlled freezing and thawing of 19 S calf thyroglobulin resulted in a specific and reproducible breakdown of the protein. Beside the elementary chain (300,000 Da), new, discrete bands are revealed by gel electrophoresis in sodium dodecyl sulfate under reducing conditions. These new species consist of a major peptide of 100,000 Da and several faster-migrating bands. Most of these polypeptides were purified by preparative gel electrophoresis and individually digested in formic acid with CNBr. A comparative gel electrophoresis under denaturating and reducing conditions of (i) the fragments obtained from the native protein, (ii) the electrophoretically purified elementary chain, (iii) the 100,000-Da peptide, and (iv) a smaller fragment (of about 50,000) was performed. It revealed a very close homology among the peptide maps of the intact 19 S, the elementary chain, and the 100,000-Da peptide. Furthermore, it was shown that the digestion products of the smaller fragment, present in the peptide map of the native protein, were absent in both the elementary chain and the 100,000-Da species. These results support the idea that calf thyroglobulin, even though it has an apparently complex molecular organization, contains structural motifs which are repeated in the elementary chain.

Iodine-induced changes in thyroglobulin half-sized subunits

The two half-sized subunits of 19 S thyroglobulin have been separated and analyzed. They share the same peptide composition and carbohydrate content. The only difference was the iodine level, which was about three times higher in the faster electrophoretic subunit.