Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

Sarcopenic Dysphagia with Low Tongue Pressure Is Associated with Worsening of Swallowing, Nutritional Status, and Activities of Daily Living.

OBJECTIVES: According to the recently proposed diagnostic criteria for sarcopenic dysphagia, sarcopenic dysphagia can be classified as probable or possible based on tongue pressure. However, it is unclear whether patients with probable and possible sarcopenic dysphagia have different characteristics. Therefore, this study aimed to investigate whether patients with possible and probable sarcopenic dysphagia have different clinical characteristics. DESIGN: A cross-sectional study. SETTING: A rehabilitation hospital. PARTICIPANTS: In total, 129 patients aged ≥65 years with sarcopenic dysphagia were included. METHODS: A tongue pressure of <20 kPa was indicative of probable sarcopenic dysphagia, and a tongue pressure of ≥20 kPa was indicative of possible sarcopenic dysphagia. Kuchi-Kara Taberu (KT) index scores were compared between the probable or possible sarcopenic dysphagia groups. RESULTS: According to the tongue pressure, 76 and 53 patients were classified into the probable and possible sarcopenic dysphagia groups, respectively. In multiple linear regression analysis, the presence of probable sarcopenic dysphagia was independently associated with the total KT index score (standardized coefficient: -0.313, regression coefficient: -4.500, 95% confidence interval [CI], -6.920 to -2.080, P < 0.001). The presence of probable sarcopenic dysphagia was independently associated with some subitems of the KT index (willingness to eat, cognitive function while eating, oral preparatory and propulsive phase, severity of pharyngeal dysphagia, eating behavior, and daily living activities). CONCLUSIONS: Patients with probable sarcopenic dysphagia were characterized by poor overall eating-related conditions, especially poor swallowing ability, ability to perform activities of daily living, and nutritional status.

Integrating 3D Rotational Angiography into Gamma Knife Planning.

3D rotational angiography provides remarkable spatial resolution for cerebrovascular disorders; however, it cannot be integrated directly into gamma knife planning due to the discrepancy of DICOM "tag" information, and most physicians still cannot benefit from 3D rotational angiography. Here, we describe a simple and easy technique to enable the integration of 3D rotational angiography.

All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution.

Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10(6) all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200,000) can leave the capsid and be replaced by water molecules from the outside in about 25 µs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

Comparative aspects on the role of polypyrimidine tract-binding protein in internal initiation of hepatitis C virus and picornavirus RNAs.

We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.

Host range of poliovirus is restricted to simians because of a rapid sequence change of the poliovirus receptor gene during evolution.

The host range of most poliovirus (PV) strains is restricted to simians. This host range specificity is believed to be determined by the interaction between PV and its receptor molecule. To elucidate the molecular basis of this species-specific infection of PV, we cloned orthologs of the PV receptor (PVR) gene ( pvr) as well as those of PV receptor-related genes 1 and 2 ( prr1 and prr2) from various mammalian species. These three genes are widely present in mammalian genomes including those of non-susceptible species. Comparison of the deduced amino acid sequences of PVR orthologs revealed that the NH(2)-terminal immunoglobulin-like domain (domain 1), which is the virus binding site in the human PVR, is highly variable among species, whereas that of PRR1 is highly conserved. Domain 1 of the PVR orthologs for the ring-tailed lemur and rabbit, which are not susceptible to PV, show only 51 and 61% amino acid sequence identity to that of human PVR, respectively. Chimeric PVR proteins that have the domain 1 of the ring-tailed lemur and rabbit PVRs failed to serve as receptors for PV. These results suggest that rapid changes in the domain 1 sequence during mammalian evolution determined the host range restriction of PV.

Molecular genetic analysis of revertants from a poliovirus mutant that is specifically adapted to the mouse spinal cord.

SA virus, a mutant of the Mahoney strain of type 1 poliovirus (PV1/Mahoney), replicates specifically in the spinal cords of mice and causes paralysis, although the PV1/Mahoney strain does not show any mouse neurovirulence (Q. Jia, S. Ohka, K. Iwasaki, K. Tohyama, and A. Nomoto, J. Virol. 73:6041-6047, 1999). The key mutation site for the mouse neurovirulence of SA was mapped to nucleotide (nt) 928 of the genome (A to G), resulting in the amino acid substitution of Met for Ile at residue 62 within the capsid protein VP4 (VP4062). A small-plaque phenotype of SA appears to be indicative of its mouse-neurovirulent phenotype. To identify additional amino acid residues involved in the host range determination of PV, a total of 14 large-plaque (LP) variants were isolated from a single point mutant, Mah/I4062M, that showed the SA phenotype. All the LP variants no longer showed any mouse neurovirulence when delivered via an intraspinal inoculation route. Of these, 11 isolates had a back mutation at nt 928 (G to A) that restored the nucleotide of the PV1/Mahoney type. The reversions of the remaining three isolates (LP8, LP9, and LP14) were mediated by a second site mutation. Molecular genetic analysis involving recombinants between Mah/I4062M and the LP variants revealed that the mere substitution of an amino acid residue at position 107 in VP1 (Val to Leu) (LP9), position 33 in VP2 (Val to Ile) (LP14), or position 231 in VP3 (Ile to Thr) (LP8) was sufficient to restore the PV1/Mahoney phenotype. These amino acid residues are located either on the surface or inside of the virus particle. Our results indicate that the mouse neurovirulence of PV is determined by the virion surface structure, which is formed by all four capsid proteins.

Recent insights into poliovirus pathogenesis.

The development of a mouse model for poliomyelitis that is transgenic for the human poliovirus receptor (hPVR) has made it much easier to investigate the efficiency of the viral dissemination process in a whole organism. These studies have given an insight into the mechanisms of blood-brain barrier permeation and neural transport. Strain-specific neurovirulence levels, however, appear to depend mainly on the replicating capacity of the virus in the central nervous system rather than the dissemination efficiency. Studies of the poliovirus-induced cytopathic effects on neural cells and specific subcellular localization of hPVR isoforms might determine a new course of investigation of poliovirus pathogenesis.

Basolateral sorting of human poliovirus receptor alpha involves an interaction with the mu1B subunit of the clathrin adaptor complex in polarized epithelial cells.

Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.

Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation.

Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H(2)O(2) or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H(2)O(2) and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H(2)O(2) or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H(2)O(2) and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H(2)O(2)-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys(598)) and second (Cys(620)) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA.

The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.

Comparison of neuropathogenicity of poliovirus type 3 in transgenic mice bearing the poliovirus receptor gene and cynomolgus monkeys.

To clarify the similarities of poliovirus infection in cynomolgus monkeys and transgenic mice bearing the poliovirus receptor, TgPVR21, we compared the pathological changes of these animals following intraspinal inoculation of two strains of poliovirus type 3 using immunohistochemical detection of the capsid antigen. All of the monkeys inoculated with 10(6) TCID(50) viruses showed flaccid paralysis 2 or 3 days post-inoculation (p.i.). TgPVR21 mice showed paralysis starting from 2 to 3 days p.i. Histologically, neurons having pyknotic nuclei and eosinophilic cytoplasm and neuronophagia were characteristically observed in both animals, but central chromatolysis was not observed in infected TgPVR21. The median lesion scores in the monkeys and TgPVR21 were well correlated, though the distribution of poliovirus-infected lesions in the central nervous system was different. In both animals the motor neurons and the brainstem nuclei responsible for flaccid paralysis were infected by the virus, while the cerebral cortex and thalamus were infected in the monkeys but not in TgPVR21. These results confirmed the reliability of neurovirulence tests using TgPVR21 as a substitute for monkeys, in respect to the spinal and brainstem lesions of poliovirus type 3.

Nuclear import of the yeast AP-1-like transcription factor Yap1p is mediated by transport receptor Pse1p, and this import step is not affected by oxidative stress.

The yeast AP-1-like transcription factor, Yap1p, is essential for the oxidative stress response in budding yeast. Yap1p is located predominantly in the cytoplasm; however, upon imposition of oxidative stress, Yap1p concentrates in the nucleus and activates target genes. Yap1p is constitutively transported in and out of the nucleus. Oxidative stress inhibits the Crm1p/Xpo1p-dependent nuclear export step, resulting in nuclear accumulation of Yap1p. In this study, we examined the mechanism for Yap1p nuclear import, and determined whether the import step is affected by oxidative stress. The nuclear accumulation of Yap1p required the activity of the small GTPase, Ran/Gsp1p. Under conditions in pse1-1 cells carrying a temperature-sensitive mutation of the importin beta family member PSE1/KAP121, nuclear translocation of Yap1p was inhibited dramatically. In an in vitro assay, we showed that Yap1p could directly bind to Pse1p and that this interaction was dissociated by Ran-GTP. These results indicate that Pse1p is the nuclear import receptor for Yap1p. In addition to Pse1p, we suggest that Kap123p, which is homologous to Pse1p, has a minor effect on the nuclear import of Yap1p. Furthermore, we identified the nuclear localization signal of Yap1p and demonstrated that the nuclear import of Yap1p was not affected by oxidative stress.

Chirality organization of ferrocenes bearing podand dipeptide chains: synthesis and structural characterization.

A variety of ferrocenes bearing podand dipeptide chains have been synthesized to form an ordered structure in both solid and solution states and have been investigated by 1H NMR, FT-IR, CD, and X-ray crystallographic analyses. Conformational enantiomerization through chirality organization was achieved by the intramolecular hydrogen bondings between the podand dipeptide chains. The single-crystal X-ray structure determination of the ferrocene 2 bearing the podand dipeptide chains (-D-Ala-D-Pro-OEt) revealed two C2-symmetric intramolecular hydrogen bondings between CO (Ala) and NH (another Ala) of each podand dipeptide chain to induce the chirality-organized structure. The molecular structures of the ferrocene 1 composed of the podand L-dipeptide chains (-L-Ala-L-Pro-OEt) and 2 are in a good mirror image relationship, indicating that they are conformational enantiomers. An opposite helically ordered molecular arrangement was formed in the crystal packing of 2 as compared with 1. The ferrocene 2 exhibited induced circular dichroism (CD), which appeared at the absorbance of the ferrocene moiety. The mirror image of the CD signals between 1 and 2 was observed, suggesting that the chirality-organized structure via intramolecular hydrogen bondings is present even in solution. The ferrocene 4 bearing the podand dipeptide chains (-Gly-L-Leu-OEt) also showed an ordered structure in the crystal based on two intramolecular hydrogen bondings between CO (Gly) and NH (another Gly) of each podand dipeptide chain, together with intermolecular hydrogen bondings between CO adjacent to the ferrocene unit and NH (neighboring Leu) to create the highly organized self-assembly. A different self-assembly was observed in the crystal of the ferrocene 5 composed of the podand dipeptide chains (-Gly-L-Phe-OEt), wherein each molecule is bonded to two neighboring molecules through two pairs of symmetrical intermolecular hydrogen bonds to form a 14-membered intermolecularly hydrogen-bonded ring. These ordered structures based on the intramolecular hydrogen bondings in the solution state are also confirmed by 1H NMR and FT-IR.

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system.

In the "selective" cholesteryl ester (CE) uptake process, surface-associated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface double-membraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

The molecular basis of poliovirus neurovirulence.

Species specificity of poliovirus (PV) is mostly governed by host cellular molecules that serve as the PV receptor (PVR). Molecular cloning of the gene and cDNAs of human PVR and the subsequent development of PV-sensitive transgenic (Tg) mice carrying the human PVR gene made it possible to investigate molecular mechanisms for PV-specific dissemination in the whole body. After intravenous inoculation which makes artificial viremia, poliovirus appears to enter the central nervous system (CNS) at a fairly high rate via the blood brain barrier, suggesting existence of a specific permeation system for PV. This main dissemination process does not require PVR. After intramuscular inoculation, PV appears to be incorporated by endocytosis at synapses, and the endosomes containing PV transported through axons to neuron cell body, where viral replication occurs. Efficiency of viral multiplication in the CNS probably determines the neurovirulence level, which differs between PV strains. An important determinant for neurovirulence phenotype resides in the internal ribosomal entry site (IRES). This has led us to a concept of "IRES-dependent virus tropism".

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.

Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis.

This study investigates the relationship between the high density lipoprotein (HDL) receptor (scavenger receptors, SR-BI and SR-BII), selective lipoprotein-cholesteryl ester uptake, and testosterone production in Leydig cells of control, hypocholesterolemic and gonadotrophic hormone (hCG) treated rats. Leydig cells from mature control rats show poor efficiency in incorporation of labeled HDL-cholesteryl esters into testosterone, poor selective uptake of lipoprotein lipids overall, and a dramatic reduction of circulating levels of lipoproteins has no apparent effect on testosterone production or expression of intracellular enzymes synthesizing cholesterol. Leydig cells from control rats show minimal levels of SR-BI and SR-BII. However, similarly aged rats treated with hCG for several days undergo changes consistent with hormone-desensitization. Despite the resulting low levels of testosterone production, SR-BI levels are dramatically increased, Leydig cells now efficiently internalize HDL-supplied cholesteryl esters by the selective cholesterol uptake process, and various other cholesterol-sensitive genes of the cells are up-regulated. Only SR-BII expression remains negligible and unchanged throughout this period. It is of interest that Leydig cell SR-BI of hCG-treated rats is localized in surface microvilli, but is present also in an elaborate and complex channel system within the cytoplasm of the cells. In summary, Leydig cells differ from other rat steroidogenic cells in not depending on exogenous lipoprotein-cholesterol during periods of normal steroid hormone production. However, trophic hormone desensitization is accompanied by increased Leydig cell SR-BI expression and increased selective HDL-cholesteryl ester uptake, presumably in preparation for renewed testosterone production.