Physiological and pathological relevance of extracellular NM23/NDP kinases

The nm23 gene is overexpressed in many hematological malignancies and other neoplasms. Some tumor cell lines that overexpress NM23 secrete this protein into extracellular environment. In this study, we found that the serum concentration of NM23-H1 protein was significantly higher in patients with various hematological malignancies. The serum level of NM23-H1 protein was clinically useful as a prognostic factor in malignant lymphoma and acute myelogeneous leukemia (AML). The level of NM23-H1 protein in all of the normal serum samples examined was lower than 10 ng/mL, while those in the tumors varied from about 0 to 1000 ng/mL. Exogenously added NM23-H1 protein did not affect the growth or survival of various leukemia and lymphoma cell lines. However, NM23-H1 protein inhibited the survival of adherent normal peripheral blood mononuclear cells (PBMNC) at 100–1000 ng/mL, and slightly stimulated the survival of nonadherent PBMNC. These results suggest that the effect of NM23-H1 protein on normal PBMNC may be associated with a poor prognosis in hematological malignancies.     

Proteomic analysis of the cyanobacterium Anabaena sp. strain PCC7120 with two-dimensional gel electrophoresis and amino-terminal sequencing

A protein-gene linkage map of the cyanobacterium Anabaena sp. strain PCC7120 was successfully constructed for 123 relatively abundant proteins. The total proteins extracted from the cell were resolved by two-dimensional electrophoresis, and the amino-terminal sequences of the protein spots were determined. By comparing the determined amino-terminal sequences with the entire genome sequence, the putative translation initiation sites of 87 genes were successfully assigned on the genome. The elucidated sequence features surrounding the translation initiation sites were as follows: (1) GTG and TTG in addition to the ATG were used as rare initiation codons; (2) the core sequences (GAGG, GGAG and AGGA) of the Shine-Dalgarno sequence were identified in the appropriate position preceding the 51 initiation sites (58.6%); (3) the nucleotides at the two regions, from -35 to -33, and from -19 to -17 (relative to the first nucleotide in the initiation codon) were preferentially adenines or thymines; (4) the nucleotides at the region from -14 to -8 were preferentially purines; (5) the nucleotide at position -1 was biased towards non-guanine (96.6%); (6) the nucleotide at the position +5 was preferentially cytosine (63.2%). It was evident that removal of the translation initiator methionine was dependent on the side-chain bulkiness of the penultimate amino acid residue. The predicted putative signal peptide sequences were also indicated. Besides confirming the existence of many predicted proteins, the data will serve as a starting point for the study of signals important in post-translational processing and nucleotide sequences important in the initiation of translation.     

Akt and PI 3-kinase signaling in cardiomyocyte hypertrophy and survival

In many systems, activation of the "protein and lipid kinase" phosphoinositide 3-kinase (PI 3-kinase) and its downstream serine-threonine kinase effector, Akt (or Protein Kinase B), provide a potent stimulus for cell proliferation, growth, and survival. In the heart, constrained by the limited proliferative capacity of cardiomyocytes, this pathway plays a key role in regulating cardiomyocyte growth and survival, with little effect on proliferation. Simultaneously, PI 3-kinase and Akt are important modulators of metabolic substrate utilization and cardiomyocyte function. Thus, the convergent signaling pathways controlling so many clinically important phenotypes of the cardiomyocyte suggest it holds promise as a therapeutic target in a variety of cardiac diseases. However, the similar role of PI 3-kinase/Akt signaling in neoplasia suggests the difficulty of activating this pathway in the heart without invoking adverse consequences elsewhere. Here we review evidence regarding the role of PI 3-kinase/Akt in controlling cardiomyocyte growth and survival, and discuss the implications for therapeutic strategies.     

Molecular mechanism of insulin resistance and obesity

Obesity and insulin resistance have been recognized as leading causes of major health issues. We have endeavored to depict the molecular mechanism of insulin resistance, focusing on the function of adipocyte. We have investigated a role of PPARgamma on the pathogenesis of Type II diabetes. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Moreover, a Pro12Ala polymorphism in the human PPARgamma2 gene was associated with decreased risk of Type II diabetes in Japanese. Taken together with these results, PPARgamma is proved to be a thrifty gene mediating Type II diabetes. Pharmacological inhibitors of PPARgamma/RXR ameliorate high-fat diet-induced insulin resistance in animal models of Type II diabetes. We have performed a genome-wide scan of Japanese Type 2 diabetic families using affected sib pair analysis. Our genome scan reveals at least 9 chromosomal regions potentially harbor susceptibility genes of Type II diabetes in Japanese. Among these regions, 3q26-q28 appeared to be very attractive one, because of the gene encoding adiponectin, the expression of which we had found enhanced in insulin-sensitive PPARgamma-deficient mice. Indeed, the subjects with the G/G genotype of SNP276 in the adiponectin gene were at increased risk for Type II diabetes compared with those having the T/T genotype. The plasma adiponectin levels were lower in the subjects with the G allele, suggesting that genetically inherited decrease in adiponectin levels predispose subjects to insulin resistance and Type II diabetes. Our work also confirmed that replenishment of adiponectin represents a novel treatment strategy for insulin resistance and Type II diabetes using animal models. Further investigation will be needed to clarify how adiponectin exerts its effect and to discover the molecular target of therapies.     

Regulation of intestinal and hypothalamic apolipoprotein A-IV

This review discusses the regulation of the intestinal and hypothalamic apolipoprotein A-IV (apo A-IV) gene and protein expression. Apo A-IV is a glycoprotein secreted together with triglyceride-rich lipoproteins by the small intestine. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. This stimulation of intestinal apo A-IV synthesis is attenuated by intravenous leptin infusion. Chronic ingestion of a high-fat diet blunts the intestinal apo A-IV in response to dietary lipid. Intestinal apo A-IV synthesis is also stimulated by members of the pancreatic polypeptide family, including peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). Recently, apo A-IV was demonstrated to be present in the hypothalamus as well. Hypothalamic apo A-IV level was reduced by food deprivation and restored by lipid feeding. Intracerebroventricular administration of apo A-IV antiserum stimulated feeding and decreased the hypothalamic apo A-IV mRNA level, implying that feeding is intimately regulated by endogenous hypothalamic apo A-IV. Central administration of NPY significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner.     

Clinical implications of thermal therapy in lifestyle-related diseases

Systemic thermal therapy, such as taking a warm-water bath and sauna, induces systemic vasodilation. It was found that repeated sauna therapy (60 degrees C for 15 min) improved hemodynamic parameters, clinical symptoms, cardiac function, and vascular endothelial function in patients with congestive heart failure. Vascular endothelial function is impaired in subjects with lifestyle-related diseases, such as hypertension, hyperlipidemia, diabetes mellitus, obesity, and smoking. Sauna therapy also improved endothelial dysfunction in these subjects, suggesting a preventive role for atherosclerosis. In animal experiments, sauna therapy increases mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in aortas. In normal-weight patients with appetite loss, repeated sauna therapy increased plasma ghrelin concentrations and daily caloric intake and improved feeding behavior. In obese patients, the body weight and body fat significantly decreased after 2 weeks of sauna therapy without increase of plasma ghrelin concentrations. On the basis of these data, sauna therapy may be a promising therapy for patients with lifestyle-related diseases.     

Analysis of complex protein-polypeptide systems for proteomic studies

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by protein extraction and characterization with chemical sequencing or mass spectrometry (MS), is the most commonly used method to analyze complex protein systems such as cells and organelles. However, it is claimed that 2-D PAGE is a slow and labor-intensive technique and also needs subsequent efforts for one-by-one identification of proteins. Recently, the combined methods of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, with preceding separation techniques such as capillary isoelectric focusing (CIEF) or liquid chromatography, have been demonstrated as high-throughput techniques suitable for proteomic analysis of protein systems. The studies which employ FTICR MS, aimed at the analysis of complex protein systems, have been reviewed, comparing their performance with that of 2-D PAGE. Also, the possibilities of combining 2-D PAGE and the FTICR MS method to analyze and reconstruct the structures and functions of complex systems are discussed.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Determination of polycyclic aromatic hydrocarbons in milk samples by high-performance liquid chromatography with fluorescence detection

This paper describes a high-performance liquid chromatographic (HPLC) method for the determination of polycyclic aromatic hydrocarbons (PAHs) in milk samples. The method involves a liquid-liquid extraction procedure after saponification of milk samples with sodium hydroxide. Reproducible determination with highly sensitive detection was attained by HPLC with fluorescence detection using 1,2-bis(9-anthryl)ethane as an internal standard. The detection limits of 12 kinds of PAHs ranged from 1.3 to 76 ng/kg milk at a signal/noise ratio of 3. By the proposed method, the presence of 12 and 11 kinds of PAHs could be confirmed in commercial milk and human milk samples, respectively. The average concentrations of total PAHs (mean+/-SD, micro g/kg) were found to be 0.99+/-0.37 for commercial milk (n=14), 2.01+/-0.30 for infant formula (n=3) and 0.75+/-0.47 for human milk (n=51). High correlation coefficients between the concentrations of total PAHs and triglyceride were observed for commercial milk (r=0.659) and human milk (r=0.645).