Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death.

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca(2+)-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2's multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.

[Bilateral multiple pulmonary arteriovenous fistulas treated by surgery and embolization].

A 46-year-old man, with an abnormal shadow in left lower area in chest X-ray was admitted to our hospital. Three dimensional computed tomography (3 D-CT) and pulmonary arteriogram revealed bilateral multiple pulmonary arteriovenous fistulas (PAVF). PAVF was simple in the right S8, and were multiple in the right lower lobe. PAVF in left side was treated with percutaneous transcatheter coil embolization. PVAFs in right side were treated by lower lobectomy. This combination therapy seemed to be useful for bilateral multiple PAVFs.

Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.

Mechanism of amelioration of insulin resistance by beta3-adrenoceptor agonist AJ-9677 in the KK-Ay/Ta diabetic obese mouse model.

The mechanism by which the specific beta3-adrenoceptor agonist AJ-9677 relieves insulin resistance in vivo was investigated by studying its effects in the white and brown adipose tissues of the KK-Ay/Ta diabetic obese mouse model. AJ-9677 reduced the total weight of white adipose tissues by reducing the size of the adipocytes, an effect associated with the normalization of tumor necrosis factor-alpha (TNF-alpha) and leptin expression levels. The levels of uncoupling protein (UCP)-1 mRNA in brown adipose tissue were increased threefold. AJ-9677 caused a marked increase (20- to 80-fold) in the expression of UCP-1 in white adipose tissues. The levels of UCP-2 mRNA were increased in both the white and brown adipose tissues of diabetic obese mice, and AJ-9677 further upregulated UCP-2 mRNA levels in brown adipose tissue, but reduced its levels in white adipose tissue. UCP-3 mRNA levels were not essentially changed by AJ-9677. However, AJ-9677 significantly (two- to four-fold) upregulated the GLUT4 mRNA and protein levels in white and brown adipose tissues and the gastrocnemius. The generation of small adipocytes, presumably mediated by increased expression of UCP-1 in addition to increased lipolysis in response to AJ-9677, was associated with decreased TNF-alpha and free fatty acid production and may be the mechanism of amelioration of insulin resistance in KK-Ay/Ta diabetic obese mice.

A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.

Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His(6)-Xpress). His(6)-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247-258, 2001)

Complete sequence and characterization of chick ventricular myosin heavy chain in the developing atria.

We isolated five complementary DNA (cDNA) clones, encoding the chick ventricular myosin heavy chain (MyHC) by reverse transcription polymerase chain reaction (RT-PCR). The entire cDNA consists of 5995 nucleotides with the 52 bp 5'-untranslated region and the 129 bp 3'-untranslated region. The complete cDNA encodes 1937 amino acids. Expression of the chick ventricular MyHC gene was also studied by Northern blot analysis. This gene continued to be strongly expressed in the ventricle during cardiac development. On the other hand, its expression was moderate in the early embryonic atria, and was down-regulated during development. In the adult atria, this gene was expressed at very low levels. To determine the localization of the ventricular MyHC protein, an immunohistochemical study was performed. The ventricular MyHC was present in early embryonic atrial myocytes. During development, the expression of this protein in the atrial myocytes was down-regulated, but continued to be present in the atrial conduction system. Our results indicate that the ventricular MyHC appears in the primary atrial myocardium and is then localized in the conduction cells of the atria.

Evidence for the expression of neonatal skeletal myosin heavy chain in primary myocardium and cardiac conduction tissue in the developing chick heart.

We isolated a neonatal skeletal myosin heavy chain (MHC) cDNA clone, CV11E1, from a cDNA library of embryonic chick ventricle. At early cardiogenesis, diffuse expression of neonatal skeletal MHC mRNA was first detected in the heart tube at stage 10. During subsequent embryonic stages, the expression of the mRNA in the atrium was upregulated until shortly after birth. It then diminished, dramatically, and disappeared in the adult. On the other hand, in the ventricle, only a trace of the expression was detected throughout embryonic life and in the adult. However, transient expression of mRNA in the ventricle was observed, post-hatching. At the protein level, during the embryonic stage, the atrial myocardium was stained diffusely with monoclonal antibody 2E9, specific for chick neonatal skeletal MHC, whereas the ventricles showed weak reactivity with 2E9. At the late embryonic and newly hatched stages, 2E9-positive cells were located clearly in the subendocardial layer, and around the blood vessels of the atrial and ventricular myocardium. These results provide the first evidence that the neonatal skeletal MHC gene is expressed in developing chick hearts. This MHC appears during early cardiogenesis and is then localized in cardiac conduction cells. Dev Dyn 2000;217:37-49.

Unique expression patterns of myosin heavy chain genes in the ductus arteriosus and uterus of rabbits.

In smooth muscle tissue, two smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2) and two non-muscle MHC isoforms (NMA, NMB) have been identified. The purpose of our study was to clarify whether smooth muscle MHC mRNA expression reflects the physiological and functional state of the muscle. We studied the expression pattern of MHC mRNAs, using the S1-nuclease mapping procedure, in functionally and morphologically changeable organs; the ductus arteriosus (DA) during development (25 and 29 days of gestation, and from 3-day-old neonates) and uteri from virgin, day-10 pregnant (P10) and day-29 pregnant (P29) rabbits. The results demonstrated that SM2 expression was greater in the fetal DA than in the fetal aortic and pulmonary arteries, but that it decreased significantly following closure of DA. In the gravid uterus, SM1 expression was significantly (P<0.05) strong compared to other MHC mRNAs from virgin to P10 rabbits. During pregnancy, NMB expression showed a tendency to increase until P10, and after P10, SM2 expression increased dramatically and NMB expression decreased to give almost a mirror image of the SM2 expression. Smooth muscle type (SM1, SM2) was significantly (P<0.05) strong compared to non-muscle type expression (NMA, NMB) at P29. These data suggest that smooth muscle MHC mRNA, especially SM2 expression reflects the physiological and functional state of the smooth muscle.

Structure and chromosomal assignment of the human lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) gene.

We have reported the cDNA cloning of a modified low-density-lipoprotein (LDL) receptor, designated lectin-like oxidized LDL receptor-1 (LOX-1), which is postulated to be involved in endothelial dysfunction and the pathogenesis of atherosclerosis. Here, we determined the organization of the human LOX-1 gene, including the 5'-regulatory region. The 5'-regulatory region contained several potential cis-regulatory elements, such as GATA-2 binding element, c-ets-1 binding element, 12-O-tetradecanoylphorbol 13-acetate-responsive element and shear-stress-responsive elements, which may mediate the endothelium-specific and inducible expression of LOX-1. The major transcription-initiation site was found to be located 29 nucleotides downstream of the TATA box and 61 nucleotides upstream from the translation-initiation codon. The minor initiation site was found to be 5 bp downstream from the major site. Most of the promoter activity of the LOX-1 gene was ascribed to the region (-150 to -90) containing the GC and CAAT boxes. The coding sequence was divided into 6 exons by 5 introns. The first 3 exons corresponded to the different functional domains of the protein (cytoplasmic, transmembrane and neck domains), and the residual 3 exons encoded the carbohydrate-recognition domain similar to the case of other C-type lectin genes. The LOX-1 gene was a single-copy gene and assigned to the p12.3-p13.2 region of chromosome 12. Since the locus for a familial hypertension has been mapped to the overlapping region, LOX-1 might be the gene responsible for the hypertension.

A mitochondrial DNA mutation cosegregates with the pathophysiological U wave.

In a family with long QT syndrome (LQT2), some individuals who did not harbor the HERG mutation had a prolonged QTU interval on electrocardiograms after exercise. It may be determined or modified by other gene(s) or factor(s). The sequence analysis of mtDNA in these individuals of this family showed a candidate pathogenic mutation at 3394 in the ND1 gene. The cybrids (mutation at 3394) showed significantly reduced NADH-CoQ reductase (complex I) activity and O2 consumption to normal levels. These inhibitory effects on respiratory function may result in the depletion of ATP and could possibly produce an increase in Ca2+ concentration in cytosol, and it may lead to the prolongation of the QTU intervals on electrocardiograms. Therefore, we stated that the 3394 mutation in the ND1 gene is pathogenic and could be the cause of prolongation of the QTU intervals or modification of the phenotypes of not only congenital but also so-called "acquired drug-induced long QT syndrome."

Paradoxical effects of pirenzepine on parasympathetic activity in chronic heart failure and control.

We studied the effect of intravenous pirenzepine (3 mg) in normal subjects (n=15, 43+/-16 years old) and in patients with chronic heart failure (n=15, 61+/-12 years old) to assess the effect of low-dose pirenzepine on vagal activity. R-R intervals and the standard deviations, low-frequency power (LF: ln ms2, 0.04-0.15 Hz), high-frequency power (HF: ln ms2, 0.15-0.40 Hz) and the ratio of low- to high-frequency power (LF/HF ratio) were measured 10 min before and after pirenzepine using a Holter analysis system. Pirenzepine was found to cause a significant increase in the R-R interval from 903+/-112 to 956+/-129 ms in the control group (P<0.0001) and from 927+/-141 to 958+/-168 ms in patients with chronic heart failure (P<0.01). Pirenzepine also increased HF significantly from 4.29+/-0.32 to 5.16+/-0.38 ln ms2 in the control group (P<0.0001) and from 4.04+/-0.16 to 4.48+/-0.24 ln ms2 in the chronic heart failure group (P<0.05). Pirenzepine did not significantly alter the LF/HF ratio in either group. We emphasize that pirenzepine appears to have a vagoinimetic effect in patients with chronic heart failure and that it may be useful for augmenting vagal control of the heart in some patients with chronic heart failure.

Evolution of the trappin multigene family in the Suidae.

Trappins are a group of secretory proteins containing a WAP motif with an anchoring domain. Previous studies showed that their genes, especially those of pig, have undergone rapid evolution, which produced trappins with a broad spectrum of actions. To understand the evolution of such a rapidly evolving multigene family, we isolated trappin genes of the Artiodactyla, including pig, wart hog, collared peccary, hippopotamus, and cow, by means of polymerase chain reaction (PCR). Two genes newly isolated from wart hog are orthologs of trappin-1 (SPAI) and trappin-2 (elafin), the others are novel members of the trappin family and named trappins-6 to 11. The divergence of the sequences is greatest in the region that encodes the reactive site, and intron sequences appear to be more highly conserved than the protein-coding sequences, especially among the pig paralogs. Phylogenetic analysis showed that the trappin multigene family members of pig were generated through gene duplication after the divergence of the Suidae (pig and wart hog) and Tayassuidae (collared peccary). Similarities in the gene structure with seminal vesicle clotting proteins (REST) and WAP motif-containing proteins suggest that trappins are naturally occurring fusion proteins created through exon shuffling between ancestral REST and WAP motif-coding genes.

Relative usefulness of measures of heart rate variability and neuroendocrine activity as indicators of autonomic nervous abnormality in patients with left ventricular dysfunction.

The aim of the study was to assess the relationship between changes in heart rate variability (HRV) and parameters of neuroendocrine activation in patients with various levels of left ventricular dysfunction. Measurements of HRV, plasma norepinephrine (PNE), and plasma atrial natriuretic peptide (ANP) were performed in 17 age- and gender-matched control subjects (group C) and in 39 patients with ischemic heart disease or cardiomyopathy who were subdivided into 3 equal groups according to their left ventricular ejection fraction: group N (normal); group M (mildly impaired); and group S (severely impaired). Spectral analysis of HRV (from 10-min electrocardiograms) was analyzed by the method of coarse-graining spectral analysis. PNE and ANP were significantly elevated in group S only (p<0.05). Log low-frequency power and log total power for group M were significantly lower than for group N (p<0.05). Log high-frequency power was significantly lower for group M than for group C (p<0.05). Thus, assessment of changes in HRV, which were observed earlier in the progress of left ventricular dysfunction than changes in neuroendocrine factors, may be a useful non-invasive method for the early detection of autonomic abnormality in patients with left ventricular dysfunction.

Association between the degree of platelet-derived growth factor-A chain mRNA expression and coronary arteriosclerosis in the transplanted heart.

Although intimal and medial proliferation of smooth muscle cells is recognized as one of the key mechanisms in the development of graft coronary arteriosclerosis, the role of platelet-derived growth factor (PDGF) in this process is still uncertain, because of the undetermined pathogenesis of graft coronary arteriosclerosis (GCA). In the present study, the correlation between the extent of GCA and the degree of PDGF-A chain expression in cardiac grafts was investigated in 21 rats with GCA of varying extent. Lewis rats underwent heterotopic heart transplantation from Wistar King donors and were treated with cyclosporine A (10 mg/kg/day) (n = 7), 15-deoxyspergualin (5 mg/kg/day) (n = 7), or Multiglycosidorum tripterygii (MT) (30 mg/kg/day) (n = 7). Histological evaluations of coronary arteriosclerosis, as well as Northern blot analysis of graft PDGF-A chain expression were made, 60 days after transplantation. Graft coronary arteriosclerosis of varying extent was observed among the 21 transplanted hearts. Significant correlations were found between the PDGF-A chain mRNA expression of cardiac allograft and the grade of arterial intimal thickening (Spearman's r = 0.76, P < 0.005) as well as the incidence of diseased vessels (r = 0.82, P < 0.001). The PDGF-A chain mRNA expression of the cardiac allograft is associated with the extent of GCA, indicating that PDGF-A plays an important role in the development of GCA.

Molecular analysis of constitutive IL-1alpha gene expression in human melanoma cells: autocrine stimulation through NF-kappaB activation by endogenous IL-1alpha.

Constitutive production of/and acquired resistance to anti-proliferative cytokines are implicated in pathogenesis and progression of human melanoma cells. Human melanoma cells A375-C6 are sensitive to interleukin 1 (IL-1) anti-proliferative effect and do not produce IL-1. After long period of culture we have obtained cells which acquired resistance to IL-1. The resistant cells exhibited constitutive production of IL-1α. To analyse the mechanisms that lead to the expression of IL-1α in the cells, we transfected of the resistant clone A375-R8 with CAT (chloramphenicol acetyltransferase) expression plasmids linked to a 5'-flanking deletion mutants of the human IL-1α gene. Two nucleotide regions (--103 to --70 bp) and (--70 to --47 bp) from the start of the first exon appeared to contain a positive regulatory element(s) while the one --421 to --103 bp contained a negative regulatory element(s).The --103 to --70 bp region contained the consensus NF(-k)B (nuclear factor-kB) binding motif.(Immunofluorescence analysis revealed that NF-kB is activated in A375-R8 cells. IL-1 receptor antagonist (IL-1Ra) decreased the level of IL-1α mRNA and production of IL-1α. IL-1Ra also inhibited the localization of p65 in the nuclei and CAT activity in transfectants with the plasmids containing NF-kB binding motif. These results indicate that endogenous IL-1α stimulates the gene expression and production of IL-1α in an autocrine manner through activation of NF-kB.

The complete sequence and expression patterns of the atrial myosin heavy chain in the developing chick.

We have earlier reported partial cloning of a cDNA of a chick atrial myosin heavy chain (MHC) gene, CCSV2 and its expression pattern in embryonic chick hearts (Oana et al (1995) Eur J Cell Biol 67, 42-49). In this study, five overlapping cDNA clones (including CCSV2) which together encode the entire open reading frame of the chick atrial MHC gene were characterized, and both the entire nucleotide sequence consisting of 5825 bases and the deduced amino acid sequence consisting of 1931 amino acids determined. Reinvestigation of the nucleotide sequence of the previously reported and presumably different chick atrial specific MHC cDNA clone, AMHC1 (Yutzey et al (1994) Development 120, 871-883), revealed that our clone and AMHC1 encoded the same MHC. The chick atrial MHC gene was strongly expressed in developing chick atria from a very early stage (Hamburger and Hamilton stage 9, 29-33 h) to the adult stage. This gene was also expressed, although weakly, in the ventricle, somite (the precursor to skeletal muscle) and skeletal muscle during embryonic stages but not in adults.