Inheritance of mitochondrial aldehyde dehydrogenase: genotyping in Chinese,Japanese and South Korean families reveals dominance of the mutant allele

Summary Genotyping of mitochondrial aldehyde dehydrogenase (ALDH I) was performed in enzymatically amplified DNA of 20 Chinese, Japanese and South Korean families (85 individuals) and in 113 unrelated persons by employing allele-specific oligonucleotide probes and dot blot hybridization. Genotyping individuals with phenotypic deficiency of ALDH I activity always showed the presence of at least one mutant allele. The data are compatible with a model assuming dominant inheritance of the mutant allele, which we have previously suggested on the basis of a population study.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as -N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of -N-methyllysines (1.40, 1.66, and 5.62 mol% for -N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of -N-methyllysines in histone H1.     

Polo-like kinase 1 (Plk1) is expressed by cutaneous T-cell lymphomas (CTCLs), and its downregulation promotes cell cycle arrest and apoptosis

Polo-like kinases are serine/threonine kinases crucial for mitosis and DNA integrity. Plk1, the most well studied member of this family, is upregulated in several cancers, as well as in dividing cells with peak expression during G2/M phase. Recently, employing lesional skin from patients with cutaneous T-cell lymphoma (CTCL), we showed that Plk1 was increased mainly in advanced lesions. In this study, employing western blot and quantitative RT-PCR analyses, we demonstrated that Plk1 was overexpressed in multiple CTCL cell lines (HH, Hut78, MyLa, SeAx and SZ4). Further, a genetic knockdown (by short hairpin RNA) or enzyme activity inhibition (via a small molecule inhibitor, GW843682X) was found to result in a decrease in cell growth, viability and proliferation. Plk1 inhibition in CTCL cells also resulted in: (1) increased G2/M phase cell cycle arrest, (2) alteration in key mitotic proteins, (3) apoptosis and (4) multiple mitotic errors. Given our findings, clinical trials of Plk1 inhibitors in CTCL may be a promising area for further translational investigation. We speculate that overexpression of Plk1 may prove to be relevant to the progression and prognosis of CTCL through its direct impact on the regulation of tumor cell proliferation and indirect influence on the acquisition of somatic mutations by proliferating tumor cells.     

The loss of phenol sulfotransferase 1 in hepatocellular carcinogenesis

Biomarkers for the detection of early hepatocellular carcinoma (HCC) are urgently needed. To identify biomarkers of HCC, we performed a comparative proteomics analysis, based on 2‐DE of HCC tissues and surrounding non‐tumor tissues. Six xenobiotic enzymes were significantly down‐regulated in the HCC tissue. Among these, phenol sulfotransferase (SULT1A1) was confirmed by Western blot analysis in 105 HCC patients. SULT1A1 showed a significant decrease in 98.1% of the HCC tissues, with 88.6% sensitivity and 66.7% specificity for the detection of HCC. Immunohistochemistry for SULT1A1 was performed and compared with glypican‐3, which is a well‐known marker of HCC. The results showed down‐regulation of SULT1A1 and up‐regulation of glypican‐3 in 52.6 and 71.9% of the HCCs, and the use of both markers improved the sensitivity up to 78.9%. Moreover, SULT1A1 was useful in differentiating early HCC from benign dysplastic nodules. Clinically, the down‐regulation of SULT1A1 was closely associated with an advanced International Union Against Cancer stage and high levels of serum α‐fetoprotein. In conclusion, the results of this study demonstrate that the loss of SULT1A1 appears to be a characteristic molecular signature of HCC. SULT1A1 might be a useful biomarker for the detection of early HCC and help predict the clinical outcome of patients with HCC.     

Studies on naturally occurring proteinous inhibitor for transmethylation reactions

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.     

Degradation of focal adhesion proteins during nocodazole-induced apoptosis in rat-1 cells

Nocodazole, a microtubule-disrupting agent, induced apoptosis in Rat-1 cells, as indicated by changes in cell morphology, DNA fragmentation, and eventual cell death. During nocodazole-induced apoptosis, normally flat cells became rounded in shape and detached from the extracellular matrix. These morphological changes appeared to be closely associated with degradation of focal adhesion proteins, including p130cas, p125(FAK) and paxillin. p130cas was also degraded in cells treated with staurosporine or etoposide, suggesting that degradation of focal adhesion proteins is a characteristic feature of apoptosis. Nocodazole-induced apoptosis was antagonized by Bcl-2: degradation of focal adhesion proteins was suppressed and cell viability was enhanced in bcl-2 over-expressing cells, even in the presence of nocodazole. Further study of the molecular mechanism of Bcl-2 activation should provide an understanding of the apoptosis induced by disruption of the microtubule network.     

Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen

Reversible phosphorylation of proteins is the most common PTM in cell‐signaling pathways. Despite this, high‐throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO₂)‐based 3‐D LC (strong cationic exchange/TiO₂/C18)‐MS³‐linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low‐abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho‐biomarkers including plectin‐1(phopho‐Ser‐4253) and alpha‐HS‐glycoprotein (phospho‐Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO₂‐based online‐3‐D LC‐MS³‐linear ion trap system with high‐throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers.