Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases

S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders.     

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

Characterization of Scale Abnormalities in Pinfish, Lagodon rhomboides, from Biscayne Bay, Florida

Correlations between marine habitat degradation and the prevalence of abnormalities and diseases in populations can provide a starting point for understanding the effects of changes in environmental conditions on marine organisms. The present study characterized the features of scale disorientation (SD), a common morphological anomaly encountered in pinfish, Lagodon rhomboides, in Biscayne Bay, Florida (U.S.A.). Scale disorientation consisted of discrete patches of scales rotated dorsally or ventrally away from the normal scale position without any projection of the scales outwards from the body surface. The direction of scale growth within the patches varied from normal to a minor misalignment to a complete reversal of direction. The severity of SD, defined as the percentage of body surface area affected, varied from 1 to 34% with a mean of 9.3%. Affected fish monitored in the laboratory demonstrated a proportional growth of SD areas such that the percentage of body surface affected did not change as the fish grew. Scale disorientation was more prevalent in the northern region of the bay, an area known to be more contaminated. Scales from SD areas exhibited significantly abnormal morphology with larger average focus diameter, smaller size, more elongate shape and fewer radii relative to normal scales. Experimental removal of scales demonstrated that normal scales regrew in normal orientation and morphology while those from SD areas regrew in abnormal orientations and morphologies. Experiments in which fish were exposed to acute and chronic injuries indicated that these physical traumas were insufficient to directly induce formation of scale disorientations typical of those seen in the wild. Observations of pinfish in the laboratory revealed that SD areas can appear spontaneously in normal juvenile and adult fish. These new SD areas developed relatively rapidly, did not require prior scale loss and remained stable in size after first appearance. Although the etiology of SD remains unknown, the significant difference in prevalence of this syndrome between regions of Biscayne Bay having different levels of sediment contaminants suggests that environmental factors may be important in development of SD.     

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

Biochemical basis for the dominant inheritance of hypermethioninemia associated with the R264H mutation of the MAT1A gene. A monomeric methionine adenosyltransferase with tripolyphosphatase activity

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PP(i). Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.     

Biochemical analysis of point mutations in the 5'-3' exonuclease of DNA polymerase I of Streptococcus pneumoniae. Functional and structural implications

To define the active site of the 5'-3' exonucleolytic domain of the Streptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His-tagged Spn pol I fusion protein and introduced mutations at residues Asp(10), Glu(88), and Glu(114), which are conserved among all prokaryotic and eukaryotic 5' nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K mutants and the control fusion protein support: (i) a critical function of Asp(10) in the catalytic event, (ii) a role of Glu(114) in the exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu(88) for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5' nucleases.     

Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.     

Sperm creatine phosphokinase activity as a measure of sperm quality in normospermic, variablespermic, and oligospermic men

We have found a significant inverse correlation between sperm concentrations and sperm creatine N-phosphotransferase (CPK) activities in oligospermic and normospermic human specimens. In the present work, we carried out serial CPK determinations to assess whether there is a relationship between fluctuating sperm concentrations and sperm quality in consistently oligospermic and variablespermic (sperm concentrations are occasionally in the greater than 20 million/ml range) husbands of 65 couples (23 normospermic men/51 samples, 25 consistently oligospermic men/80 samples, and 17 variablespermic men/68 samples). The sperm CPK activities were significantly lower in the normospermic vs. the oligospermic or variablespermic groups (p less than 0.001), but there were no differences between the latter two (p greater than 0.25). The mean CPK values of migrated sperm fractions in both the oligospermic and variablespermic populations were improved (at least 20% decline in CPK values) compared to those of the initial specimens (1.27 +/- 0.38 vs. 0.68 +/- 0.37 and 0.77 +/- 0.32 vs. 0.46 +/- 0.24 SEM U/100 million sperm, respectively, p less than 0.001 in both pairs) and the incidence of the "failed-to-improve" samples was also similar in the two groups (44/36 vs. 45/23, p greater than 0.2). The lack of differences in the mean CPK activities, in the distribution of CPK values under and over 0.250 U/100 million sperm level, and in the ratio of migrated samples with improved or with failed-to-improve CPK activities suggests that sperm quality is not different between men who are consistently oligospermic and those who occasionally produce normospermic specimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatography determination of glyoxal,methylglyoxal, and diacetyl in urine using 4-methoxy-o-phenylenediamine as derivatizing reagent

Bioanalytical relevance of glyoxal (Go) and methylglyoxal (MGo) arises from their role as biomarkers of glycation processes and oxidative stress. The third compound of interest in this work is diacetyl (DMGo), a component of different food products and alcoholic beverages and one of the small α-ketoaldehydes previously reported in urine. The original idea for the determination of the above compounds by reversed phase high-performance liquid chromatography (HPLC) with fluorimetric detection was to use 4-methoxy-o-phenylenediamine (4MPD) as a derivatizing reagent and diethylglyoxal (DEGo) as internal standard. Acetonitrile was added to urine for matrix precipitation, and derivatization reaction was carried out in the diluted supernatant at neutral pH (40 °C, 4 h); after acidification, salt-induced phase separation enabled recovery of the obtained quinoxalines in the acetonitrile layer. The separation was achieved within 12 min using a C18 Kinetex column and gradient elution. The calibration detection limits for Go, MGo, and DMGo were 0.46, 0.39, and 0.28 μg/L, respectively. Within-day precision for real-world samples did not exceed 6%. Several urine samples from healthy volunteers, diabetic subjects, and juvenile swimmers were analyzed. The sensitivity of the procedure proposed here enabled detection of differences between analyte concentrations in urine from patients at different clinical or exposure-related conditions.