Effects of resistance training on arterial compliance and plasma endothelin-1 levels in healthy men.

Arterial compliance (AC) is an index of the elasticity of large arteries. Endothelial dysfunction has been reported to result in reduced arterial compliance, which represents increased arterial stiffness. A reduction in AC is elicited by high-intensity resistance training, however the mechanisms are obscure. Because a single bout of resistance exercise causes a transient increase in circulating plasma endothelin-1 in humans, some vasoconstrictors may play a role in the mechanisms. The present study aimed to investigate whether resistance training-induced decrease in AC is associated with changes in circulating vasoconstrictors levels in young men. Young sedentary men were assigned to control (n=5) or training (n=9) groups. The training group performed four-week high-intensity resistance training (weight training exercise; three sessions/week). We measured AC and plasma levels of endothelin-1, angiotensin II, and norepinephrine before and after intervention. Resistance training significantly decreased AC, whereas the changes in plasma levels of neither endothelin-1, nor angiotensin II, nor norepinephrine were significantly different between the control and the training groups. Moreover, we found no significant correlations between changes in circulating plasma levels (endothelin-1, angiotensin II, and norepinephrine) and in the AC. Despite of no alteration of the resting circulating plasma levels (endothelin-1, etc.), we cannot exclude a possibility that the tissue/local concentrations of vasoconstrictors (endothelin-1, etc.) around the vessels might be increased and also involved in a reduction of AC in the training group. Taken together, the present results suggest that circulating vasoconstrictors (endothelin-1, etc.) in plasma are not involved in a reduction in AC by the resistance training.

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

Test validity of periodic liver function tests in a population of Japanese male bank employees.

The validity (sensitivity and specificity) of annual liver function tests, determined by assaying blood levels of aspartate aminotransferase, alanine aminotransferase and gammaglutamyl transpeptidase, was evaluated using the results of health checkups of male bank workers. The specificity of each liver function test to detect persons with fatty liver, excess alcohol users, and hepatic virus carriers, diagnosed respectively by ultrasound, detailed inquiry, and virus marker tests, was always higher than 80%, except for alanine aminotransferase in excess alcohol users (63.5%). However, the highest sensitivity to detect virus carriers was alanine aminotransferase to detect HCV antibody-positive workers, but it was only 45.5%. The highest sensitivity of the liver function tests to detect excess alcohol users in obese subjects was only 33.3%. The highest sensitivity by liver function tests to detect fatty liver was 35.7% which was inferior to that of the body mass index. These results indicate that the liver function tests mandated in the workplace periodic health checkups in Japan exhibit very low sensitivity for the detection of any of the proposed target clinical conditions.

Selective deficiency of alpha-dystroglycan in Fukuyama-type congenital muscular dystrophy.

BACKGROUND: Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by severe dystrophic muscle wasting from birth or early infancy with structural brain abnormalities. The gene for FCMD is located on chromosome 9q31, and encodes a novel protein named fukutin. The function of fukutin is not known yet, but is suggested to be an enzyme that modifies the cell-surface glycoprotein or glycolipids. OBJECTIVE: To elucidate the roles of fukutin gene mutation in skeletal and cardiac muscles and brain. METHODS: Immunohistochemical and immunoblot analyses were performed in skeletal and cardiac muscles and brain tissue samples from patients with FCMD and control subjects. RESULTS: The authors found a selective deficiency of highly glycosylated alpha-dystroglycan, but not beta-dystroglycan, on the surface membrane of skeletal and cardiac muscle fibers in patients with FCMD. Immunoblot analyses also showed no immunoreactive band for alpha-dystroglycan, but were positive for beta-dystroglycan in FCMD in skeletal and cardiac muscles. CONCLUSION: The current findings suggest a critical role for fukutin gene mutation in the loss or modification of glycosylation of the extracellular peripheral membrane protein, alpha-dystroglycan, which may cause a crucial disruption of the transmembranous molecular linkage of muscle fibers in patients with FCMD.

Production of acetate in the liver and its utilization in peripheral tissues.

In experimental rat liver perfusion we observed net production of free acetate accompanied by accelerated ketogenesis with long-chain fatty acids. Mitochondrial acetyl-CoA hydrolase, responsible for the production of free acetate, was found to be inhibited by the free form of CoA in a competitive manner and activated by reduced nicotinamide adenine dinucleotide (NADH). The conditions under which the ketogenesis was accelerated favored activation of the hydrolase by dropping free CoA and elevating NADH levels. Free acetate was barely metabolized in the liver because of low affinity, high K(m), of acetyl coenzyme A (acetyl-CoA) synthetase for acetate. Therefore, infused ethanol was oxidized only to acetate, which was entirely excreted into the perfusate. The acetyl-CoA synthetase in the heart mitochondria was much lower in K(m) than it was in the liver, thus the heart mitochondria was capable of oxidizing free acetate as fast as other respiratory substrates, such as succinate. These results indicate that rat liver produces free acetate as a byproduct of ketogenesis and may supply free acetate, as in the case of ketone bodies, to extrahepatic tissues as fuel.

Recognition of novel lipopolypeptides with many pendent sugar residues by lectin.

A lipid-polypeptide conjugate (lipo-polypeptide) was obtained by the ring-opening polycondensation of N-epsilon-Z-L-lysine N-carboxyanhydride (NCA) using 3-aminopropyl dioctadecylamine as initiator and subsequent deprotection. Maltose lactone was coupled with the lipo-polypeptide to give novel amphiphiles which carried many maltoamide residues as pendent groups. The sugar group-carrying amphiphiles incorporated in phospholipid liposomes were recognized by a lectin from Canavalia ensiformis (Con A), which was proven by the increase in turbidity of the liposome suspension after mixing with the lectin. The recognition was largely affected by the degree of polymerization of lysine residues and the surface density of the amphiphile in the liposomes. The association constant (K(ass)) of Con A with maltoamide residues on the liposome was much larger than those for small molecular weight sugars due to the "cluster effect".

Deuterostome evolution: early development in the enteropneust hemichordate, Ptychodera flava.

Molecular and morphological comparisons indicate that the Echinodermata and Hemichordata represent closely related sister-phyla within the Deuterostomia. Much less is known about the development of the hemichordates compared to other deuterostomes. For the first time, cell lineage analyses have been carried out for an indirect-developing representative of the enteropneust hemichordates, Ptychodera flava. Single blastomeres were iontophoretically labeled with Dil at the 2- through 16-cell stages, and their fates followed through development to the tornaria larval stage. The early cleavage pattern of P. flava is similar to that of the direct-developing hemichordate, Saccoglossus kowalevskii, as well as that displayed by indirect-developing echinoids. The 16-celled embryo contains eight animal "mesomeres," four slightly larger "macromeres," and four somewhat smaller vegetal "micromeres." The first cleavage plane was not found to bear one specific relationship relative to the larval dorsoventral axis. Although individual blastomeres generate discrete clones of cells, the appearance and exact locations of these clones are variable with respect to the embryonic dorsoventral and bilateral axes. The eight animal mesomeres generate anterior (animal) ectoderm of the larva, which includes the apical organ; however, contributions to the apical organ were found to be variable as only a subset of the animal blastomeres end up contributing to its formation and this varies from embryo to embryo. The macromeres generate posterior larval ectoderm, and the vegetal micromeres form all the internal, endomesodermal tissues. These blastomere contributions are similar to those found during development of the only other hemichordate studied, the direct-developing enteropneust, S. kowalevskii. Finally, isolated blastomeres prepared at either the two- or the four-cell stage are capable of forming normal-appearing, miniature tornaria larvae. These findings indicate that the fates of these cells and embryonic dorsoventral axial properties are not committed at these early stages of development. Comparisons with the developmental programs of other deuterostome phyla allow one to speculate on the conservation of some key developmental events/mechanisms and propose basal character states shared by the ancestor of echinoderms and hemichordates.

Molecular studies of hemichordate development: a key to understanding the evolution of bilateral animals and chordates.

Using the Hawaiian acorn worm, Ptychodera flava, we began molecular studies on the development of hemichordates, a phylum previously unstudied at this level. Here we review results garnered from the examination of a few specific genes selected to help understand the evolution of vertebrate structures. These studies suggest new ideas about the evolution of developmental mechanisms in the deuterostomes. In a seminal observation, we noted an unexpected zone of expression of the Brachyurygene in the early anterior embryonic ectoderm where the mouth will form. Typically, the Brachyury gene is closely linked to development of the notochord and is expressed around the blastopore and in the posterior mesoderm in most animals. This first expression of Brachyury at the blastopore may represent a regulatory program associated with organizing the original animal head and gut opening, as suggested by the expression of Brachyury during hypostome formation in hydra. We believe that the anterior expression of Brachyury in deuterostomes represents the cooption of the program for organizing the original animal gut opening to form the deuterostome mouth. Recent data from the trochophore larva of a polychaete show that an anterior zone of expression of Brachyury is produced in this protostome by splitting of the Brachyury field during the formation of a gut with a mouth and anus by the lateral fusion of the sides of the blastopore. The ability to initiate independently a secondary regulatory program to organize the new mouth leading to an anterior field of Brachyury expression may be a signal event in the evolution of the deuterostomes. We also noted that the P. flava homolog of T-brain/Eomes, a gene closely related by sequence and expression around the blastopore to Brachyury and associated with development of the vertebrate brain, also exhibits early posterior expression around the blastopore and a field of de novo anterior ectoderm expression during later embryogenesis. The tissue in the zone of de novo anterior ectoderm expression of Pf-Tbrain produces the apical organ, a larval neural structure that has been touted as an evolutionary precursor of the chordate dorsal brain. The gene regulatory mechanisms responsible for initiating the anterior zone of de novo expression of T-brain may represent a cooption to specify early neuroectoderm of the regulatory program evolved first to drive anterior Brachyury expression for deuterostome mouth formation. It will be interesting to examine the possibilities that an ability to initiate the de novo anterior expression of the program that includes T-brain may be a key event in the evolution of the developmental mechanisms leading to the chordate dorsal nervous system.

Correlation between genetic alterations and histopathological subtypes in bronchiolo-alveolar carcinoma and atypical adenomatous hyperplasia of the lung.

Bronchiolo-alveolar carcinoma (BAC) is a type of lung adenocarcinoma characterized by growth along the alveolar wall. It is divided into two subtypes: sclerosing BAC (SBAC), which has central fibrosis, and non-sclerosing BAC (NSBAC), which lacks central fibrosis. We compared the genetic alterations in these two types of BAC with those in atypical adenomatous hyperplasia (AAH). There were 39 cases of SBAC, 19 of NSBAC and 20 of AAH. To detect the loss of heterozygosity (LOH) we used the microsatellite markers D3S1234 and D3S1300 on chromosome 3p, IFNA and D9S144 on 9p, and TP53 on 17p. We also used polymerase chain reaction-SSCP analysis and direct sequencing to examine a point mutation of the p53 gene at exons 5-8. At the TP53 locus, the frequencies of LOH showed a statistical rank-difference correlation among AAH, NSBAC and SBAC. On chromosomes 3p and 9p there were no statistical differences of LOH among AAH, NSBAC and SBAC. We detected a significant statistical rank-difference correlation in the p53 mutation among AAH, NSBAC and SBAC. These findings suggest that a process of multistep carcinogenesis from AAH through NSBAC to SBAC might occur in some cases of adenocarcinoma, and LOH of 3p and 9p might be an early event of carcinogenesis, while the p53 mutation might be a later event.

Correlation between morphological heterogeneity and genetic alteration within one tumor in adenocarcinomas of the lung.

In some human cancers, multistep carcinogenesis has been advocated on the basis of morphological and genetic analysis. In adenocarcinoma of the lung, a carcinogenetic process from atypical adenomatous hyperplasia (AAH) to bronchiolo-alveolar carcinoma (BAC) and/or more malignant adenocarcinoma has been recently suggested. In the present study, we selected 13 lung tumors which had AAH-like or BAC-like areas at the periphery, and poorly differentiated areas at other sites, and examined their loss of heterozygosity (LOH) on chromosome 3p, 9p and 17p and point mutation of the p53 gene. A heterogeneous pattern of LOH and/or point mutation of the p53 gene was detected in five of 13 cases, and genetic alterations were frequent in the areas of poorer differentiation. These findings suggest that some adenocarcinomas of the lung occur through multistep carcinogenesis.

Auditory agnosia restricted to environmental sounds following cortical deafness and generalized auditory agnosia.

We encountered a case of auditory agnosia restricted to environmental sounds, which was associated with the development of bilateral subcortical lesions after suffering a bilateral putaminal hemorrhage. The patient had a history of a putaminal hemorrhage on her left side without any major disability. Three years later, she suffered a putaminal hemorrhage on the other side. The clinical picture started with cortical deafness, then changed to generalized auditory agnosia for verbal and environmental sounds, and finally developed into auditory agnosia confined to the perception of environmental sounds. Her errors in a test of sound recognition were discriminative rather than associative in nature. Neuro-radiological examinations revealed bilateral subcortical lesions involving the fibers from the medial geniculate body to the temporal lobes after bilateral putaminal hemorrhage. This case suggested that the subcortical lesion involving bilateral acoustic radiation could cause either cortical deafness, auditory agnosia of all sounds, or auditory agnosia restricted to environmental sounds.

Deep white matter lesions on MRI, and not silent brain infarcts are related to headache and dizziness of non-specific cause in non-stroke Japanese subjects.

OBJECTIVE: Silent or asymptomatic cerebrovascular disease is believed to be an important risk factor for symptomatic stroke and vascular dementia. Although non-specific complaints such as mild to moderate headache and/or dizziness may also be caused by silent stroke, which remains a topic of controversy. METHODS: To investigate the relationship between silent brain infarcts and non-specific complaints, we assessed findings on magnetic resonance images using a common protocol in the following three groups of subjects; Group 1:78 subjects with non-specific complaints, Group 2:47 subjects with vascular risk factors, and Group 3:75 normal subjects without any subjective complaints or vascular risk factors. In addition to silent stroke, deep white matter lesions on MRI were also evaluated. All subjects were recruited from 12 institutes of the study group located at various parts of Japan. RESULTS: Silent brain infarcts were demonstrated in 44%, 43%, and 20% of subjects in Groups 1, 2, and 3, respectively. In Group 1, the average number of infarcts per individual who had silent brain infarction was 1.8, which was significantly fewer than 3.8 in Group 2 or 3.5 in Group 3 (p<0.0167). White matter lesions were found in 68%, 49%, and 11% in Groups 1, 2, and 3, respectively, indicating that non-specific complaints are more closely related to deep white matter lesions than to silent infarct lesions. Such white matter lesions were found more frequently in subjects with depressive state than in non-depressed subjects (67% vs. 39%, p=0.0155). CONCLUSION: The present results suggest that deep white matter lesions, rather than silent brain infarcts, appear to be important in producing headache and/or dizziness of non-specific cause and also to be related to the depressive state.

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A.

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.

T-Brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain.

T-box genes encode a novel family of sequence-specific activators that appear to play crucial roles in various processes of animal development. Although most of the T-box genes are involved in the mesoderm formation of chordate embryos, mammalian T-Brain is expressed in the developing central nervous system, and defines molecularly distinct domains within the cerebral cortex. Here we report the first invertebrate T-Brain homologue from the hemichordate acorn worm, Ptychodera flava, which we designate Pf-Tbrain. Developmental expression of Pf-Tbrain was examined by whole mount in situ hybridization to various stages of P. flava embryos. A weak, broad in situ hybridization signal of the Pf-Tbrain transcript is first detected during gastrulation in cells around the archenteron, but this signal disappears as gastrulation proceeds. At mid-gastrula an intense signal appears in several apical ectoderm cells of the gastrula. This signal becomes restricted to the apical region, where the eyespots or the light-sensory organ of the tornaria larva form. Expression of Pf-Tbrain in the apical sensory organ of the tornaria and vertebrate T-Brain in the forebrain suggests an evolutionary relationship between the non-chordate deuterostome larval apical sensory organ and the chordate forebrain.

Developmental expression of the hemichordate otx ortholog.

The phylogenetic location of hemichordates is unique because they seem to fill an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva.

Characterization of gill-specific genes of the acorn worm Ptychodera flava.

Acorn worms are hemichordate deuterostomes that have remarkable gills thought to be homologous to pharyngeal gills in urochordates and cephalochordates, and pharyngeal pouches in vertebrates. In search of molecular keys to analyzing the origin and evolution of the anterior gut and neck region of the chordate body, the present study isolated cDNA clones for six gill-specific genes, designated PfG1 to PfG6, from Ptychodera flava using differential screening of a cDNA library of RNA from gills. Northern blotting confirmed that these genes were all expressed only in the gills. In situ hybridization showed that the expression of these genes is limited to the endodermally derived columnar epithelium of the pharynx. PfG1 encodes a 42-kDa polypeptide containing sequence similar to D-domains, protein domains characteristic of extracellular proteins. Expression of PfG1 is localized in a delimited pattern along the columnar epithelium of the inner gill apparatus. Expression in the epibranchial ridge appears as two stripes running longitudinally in the epithelium just lateral of the midline. A stripe of expression also appears in a slightly posterior portion on the curve of each band of columnar epithelium on the pharyngeal surface of the secondary gill bars. The five other gill-specific genes, PfG2 to PfG6, encode a family of C-type lectin polypeptides that appear to be secreted proteins. PfG2 to PfG6 are also expressed in the columnar epithelium of the epibranchial ridge as two parallel stripes, but at the lateral margin of the ridge. One of the genes, PfG6, is additionally expressed in the innermost curve of the epithelium on the pharyngeal surface of each secondary gill bar. The localization of expression of PfPax1/9, a gill-specific transcription factor gene, was examined and shown to also be primarily in the endodermal columnar epithelium on the pharyngeal faces of the gill bars. On the secondary gill bars, where PfG1 and PfG6 are also expressed in the columnar epithelium, PfPax1/9 is expressed in the anterior and posterior portions but signal is not evident in the epithelium on the central, innermost curve of the gill bar. The anterior domain of PfPax1/9 expression is more extensive but overlaps the anterior domain of PfG1 expression, whereas its posterior domain of expression is more posterior and complementary to that of PfG6.

Characterization of a hemichordate fork head/HNF-3 gene expression.

Based on anatomical and developmental similarities, hemichordates are thought to be most closely related to chordates. However, so far very few developmental genes have been characterized from hemichordates. To gain molecular insight into the developmental mechanisms involved in the origin and evolution of chordates, we investigated the expression of a fork head/HNF-3 (PfHNF3) gene in the acorn worm embryo. Chordate fork head genes are implicated in the formation of endoderm, notochord and floor plate. We found that a PfHNF3 transcript was first detected at the early blastula stage; the signal of in situ hybridization was found in the vegetal plate cells, invaginating endoderm and then in the archenteron. By the late gastrula and into the early tornaria larva stages, an intense signal remained in the anterior region of the archenteron, while the expression in the other regions of archenteron decreased. The intense signal was retained in the pharynx of the tornaria larva. A comparison of the pattern of PfHNF3 with that of HNF-3 genes of sea urchin, ascidian, amphioxus and vertebrate suggests a possible acquisition of new functions of the gene during deuterostome evolution.

Down-regulation of KAI1 messenger RNA expression is not associated with loss of heterozygosity of the KAI1 gene region in lung adenocarcinoma.

KAI1, a metastasis suppressor gene of prostate cancer, is located on human chromosome 11p11.2. Down-regulation of KAI1 mRNA during tumor progression and metastasis has been reported for several kinds of cancer, but the mechanism of this down-regulation is not known. In the present study, our aim was to ascertain the relationship between down-regulation of KAI1 mRNA expression and KAI1 gene alterations in lung cancer. Forty-nine cases of adenocarcinoma of the lung were studied by reverse-transcriptase polymerase chain reaction (RT-PCR) assay of KAI1 mRNA and by immunohistochemical detection of KAI1 protein. In addition, markers of the microsatellite loci D11S1344 and D11S1326 were used to investigate loss of heterozygosity (LOH) and replication errors (RERs) of the KAI1 gene region. The RT-PCR assay showed that there was no correlation between KAI1 mRNA expression and either the age of the patients or tumor size. By contrast, KAI1 mRNA expression was significantly correlated with gender (P=0.047), metastasis to the lymph nodes or other organs (P=0.004), the histological grade of the tumor (P=0.036) and the pathological stage (P=0.049). Immunohistochemical staining showed that in one case without metastasis, loss of KAI1 mRNA was associated with invasion of the stroma by KAI1 protein-negative cancer cells. The numbers of informative cases by microsatellite analysis were 14 (28.6%) of 49 at D11S1344 and 27 (55.1%) of 49 at D11S1326; none of 49 adenocarcinomas showed LOH or RERs at these loci. These results suggest that down-regulation of KAI1 mRNA expression rarely if ever involves LOH or RERs of the KAI1 gene region in primary lung adenocarcinoma.

Balloon-occluded retrograde transvenous obliteration of gastric varix draining via the left inferior phrenic vein into the left hepatic vein.

We encountered a patient with gastric varix draining not via the usual left suprarenal vein but via the left inferior phrenic vein joining the left hepatic vein. Transfemoral balloon-occluded retrograde transvenous obliteration (BRTO) of the varix was performed under balloon occlusion of the left inferior phrenic vein via the left hepatic vein and retrograde injection of the sclerosing agent (5% of ethanolamine oleate) into the gastric varix. Disappearance of the gastric varix was confirmed on endoscopic examination 2 months later.