Increased plasma HB-EGF associated with obesity and coronary artery disease

The mechanism by which the obese subjects are more associated with vascular disease remains unclear. We reported that the adipose tissues produce and secrete many bioactive molecules, conceptualized as adipocytokines. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), produced locally by vascular macrophages and smooth muscle cells, has been suggested to induce the migration and proliferation of vascular smooth muscle cells. The current study reveals that (1) HB-EGF mRNA is abundantly expressed in human adipose tissue, (2) HB-EGF mRNA increases in the fat tissues of obese mice, (3) plasma HB-EGF levels increase in parallel with fat accumulation in human, and (4) the subjects with coronary artery disease have higher plasma HB-EGF levels, associated with fat accumulation. These results suggest that increased plasma HB-EGF derived from the accumulated fat contributes to the higher incidence of vascular disease in obesity, proposing HB-EGF as an adipocytokine directly linking adipovascular axis.     

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.     

Normal telomere lengths of spermatozoa in somatic cell-cloned bulls

Interesting questions have been raised regarding cloned animals, including whether cloning restores cellular senescence undergone by donor cells, and how long cloned animals will be able to live. In this study, focusing our attention on the fact that telomere lengths of spermatozoa are longer than those of any somatic cells and that telomere length is maintained throughout aging in humans, we compared the telomere lengths of spermatozoa in normal and two somatic cell-cloned cattle. The telomere lengths of the spermatozoa in the normal cattle (22.42+/-0.32 kb) were maintained throughout aging as in humans. In the cloned cattle, telomere lengths of the spermatozoa (25.8 and 20.9 kb) were the same as or longer than those found in normal cattle. Considering that telomere lengths of the donor cells, which had been derived from the muscle tissue of an old bull, were reported to be 20.1 kb, the results suggested that the telomere lengths of the germ cell line had extended from nucleus transfer to spermatogenesis. Moreover, we produced offspring (nine calves) from a somatic cell-cloned bull and measured the telomere lengths of their leukocytes. In all of the offspring, the telomere lengths of leukocytes were normal, too. These results indicate the possibility that somatic cloned bulls could be used as breeding sires.     

Upregulation of redox-regulating protein,thioredoxin, in endomyocardial biopsy samples of patients with myocarditis and cardiomyopathies

An important role of redox regulation in myocardial diseases and heart failure has been postulated. Thioredoxin (TRX) is a redox-regulating protein. Recent studies indicated a possible association between plasma TRX concentrations and the severity of heart failure. Accordingly, we investigated the myocardial expression of TRX in patients with myocarditis and cardiomyopathies. Four cases of hypertrophic cardiomyopathy (HCM), 10 of dilated cardiomyopathy (DCM), 6 of myocarditis, and 5 of controls were studied. Right and left ventricular endomyocardial biopsy samples were obtained at the diagnostic cardiac catheterization. The samples were processed for immunohistological staining for TRX, which was done by the indirect immunoperoxidase technique. 8-hydoxy-2-deoxyguanosine (8-OHdG), one of the major DNA base-modified products, was also detected for an established marker for oxidative stress. TRX immunoreactivity was none or trivial in control specimens. Positive TRX staining was found in 6 cases; 3 in active myocarditis and 3 in DCM. The positive staining was found in infiltrating cells and damaged myocytes in the perinecrotic lesions. Damaged myocytes were also positive for 8-OHdG. All the 3 cases of DCM positive for TRX stain showed severe left ventricular hypertrophy on electrocardiogram and highly elevated left ventricular end-diastolic pressure (> 24 mmHg), suggesting the overload of oxidative stress by hemodynamic impairment. Myocardial TRX was upregulated in myocarditis and cardiomyopathies with active necrotic stage associated with DNA damage, which may reflect the oxidative stress overload in hemodynamically uncontrolled status.     

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.     

Amyloid fibril formation by the CAD domain of caspase-activated DNase

Caspase-activated DNase (CAD) is a key protein in the process of apoptosis that degrades DNA through the action of caspases. Its N-terminal region, the CAD domain (CAD-CD), is highly conserved among CAD family proteins and is responsible for the interaction with its inhibitor. We report here that CAD-CD spontaneously aggregates to form amyloid fibrils, without a lag time, under the conditions of low pH (below 4) and the presence of anions. Interestingly, the secondary structure of CAD-CD in the fibril state comprised not only beta-sheet but also alpha-helix, as found in CD, FTIR, and x-ray fiber diffraction experiments. Aromatic side chains have a defined orientation and are in the hydrophobic environment occurring with the CAD-CD fibrillogenesis. These findings provide new insights into the architecture of amyloid fibrils.     

Self-organizing-map-based molecular signature representing the development of hepatocellular carcinoma

Using high-density oligonucleotide array, we comprehensively analyzed expression levels of 12600 genes in 50 hepatocellular carcinoma (HCC) samples with positive hepatitis C virus (HCV) serology (well (G1), moderately (G2), and poorly (G3) differentiated tumors) and 11 non-tumorous livers (L1 and L0) with and without HCV infection. We searched for discriminatory genes of transition (L0 vs. L1, L1 vs. G1, G1 vs. G2, G2 vs. G3) with a supervised learning method, and then arranged the samples by self-organizing map (SOM) with the discriminatory gene sets. The SOM arranged the five clusters on a unique sigmoidal curve in the order L0, L1, G1, G2, and G3. The sample arrangement reproduced development-related features of HCC such as p53 abnormality. Strikingly, G2 tumors without venous invasion were located closer to the G1 cluster, and most G2 tumors with venous invasion were located closer to the G3 cluster (P=0.001 by Fisher's exact test). Our present profiling data will serve as a framework to understand the relation between the development and dedifferentiation of HCC.     

Repetitive interactions observed in the crystal structure of a collagen-model peptide, [(Pro-Pro-Gly)9]3

The crystal structure of a collagen-model peptide [(Pro-Pro-Gly)(9)](3) has been determined at 1.33 A resolution. Diffraction data were collected at 100 K using synchrotron radiation, which led to the first structural study of [(Pro-Pro-Gly)(n)](3) under cryogenic conditions. The crystals belong to the P2(1) space group with cell parameters of a = 25.95, b = 26.56, c = 80.14 Angstroms and beta = 90.0 degrees. The overall molecular conformation was consistent with the left-handed 7/2-helical model with an axial repeat of 20 A for native collagen. A total of 332 water molecules were found in an asymmetric unit. Proline residues in adjacent triple-helices exhibited three types of hydrophobic interactions. Furthermore, three types of hydrogen-bonding networks mediated by water molecules were observed between adjacent triple-helices. These hydrophobic interactions and hydrogen-bonding networks occurred at intervals of 20 Angstroms along the c-axis based on the previous sub-cell structures [(Pro-Pro-Gly)(n)](3) (n = 9, 10), which were also seen in the full-cell structure of [(Pro-Pro-Gly)(10)](3). Five proline residues at the Y position in the X-Y-Gly triplet were found in a down-puckering conformation, this being inconsistent with the recently proposed propensity-based hypothesis. These proline residues were forced to adopt opposing puckering because of the prevailing hydrophobic interaction between triple-helices compared with the Pro:Pro stacking interaction within a triple-helix.     

An unusual 2D → 3D parallel interpenetration: synthesis and X-ray structure of compound [Ag2(titmb)2][Hsal]2 · 3H2O (titmb=1,3,5-tris(imidazol-1-ylmethyl)-2,4,6-trimethylbenzene and H2sal=salicylic acid)

The reaction between 1,3,5-tris(imidazol-1-ylmethyl)-2,4,6-trimethylbenzene (titmb) and [Ag(NH₃)₂][Hsal] (H₂sal=salicylic acid) produces novel complex [Ag₂(titmb)₂][Hsal]₂ · 3H₂O (1). There are two unique silver centers in complex 1 with one serving as a trigonal node and the other one as a linear node. Interestingly, there are two sorts of titmb ligands in complex 1, one acting as a three-connecting ligand and the other one as a two-connecting (bridging) ligand. The silver centers are linked by titmb ligand to form 2D puckered layers containing M₅(titmb)₅ macrocyclic motifs, which is large enough to allow the bridging titmb ligands from the neighboring layers to be completely enclosed in. Namely, two neighboring independent layers interpenetrated each other in a parallel fashion without the occurrence of catenation to give a 3D structure.     

Structure-activity relationships of a novel class of endothelin receptor selective antagonists; 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridines

The synthesis and structure-activity relationships of 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridine class of ET(A) receptor selective antagonists were described. These derivatives were prepared from the optically active key intermediates (3, 4, 10, and 13). Optimization of the substituent at the 2-position of the bottom 4-methoxyphenyl ring of the lead compound 1 led to identification of 2-hydroxy-1-methylethoxy (2g and h), hydroxyalkyl (2i, m, and p), 3-methoxy-2-methylpropyl (2t and u), N-acetyl-N-methylaminomethyl (2v), and 2-(dimethylcarbamoyl)propyl (2w) derivatives that showed greater than 1000-fold selectivity for the ET(A) receptor over the ET(B) receptor with excellent binding affinity (IC(50)<0.10 nM). Further screening of these compounds by assessing the plasma exposures at 1 h, 4 h, and 8 h after oral administration (3 or 10 mg/kg) in rats led to identification of the hydroxymethyl (2i) and 3-methoxy-2-methylpropyl (2u) derivatives exhibiting good oral bioavailability in rats.