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Background: Cell population data (CPD) parameters related to neutrophils, such as fluorescent light intensity (NE-SFL) and fluorescent light distribution width index (NE-WY), have emerged as potential biomarkers for sepsis. However, the diagnostic implication in acute bacterial infection remains unclear. This study assessed the diagnostic value of NE-WY and NE-SFL for bacteremia in patients with acute bacterial infections, and those associations with other sepsis biomarkers., Methods: Patients with acute bacterial infections were enrolled in this prospective observational cohort study. For all patients, a blood sample, with at least two sets of blood cultures, were collected at the onset of infection. Microbiological evaluation included examination of the blood bacterial load using PCR. CPD was assessed using Automated Hematology analyzer Sysmex series XN-2000. Serum levels of procalcitonin (PCT), interleukin-6 (IL-6), presepsin, and CRP were also assessed., Results: Of 93 patients with acute bacterial infection, 24 developed culture-proven bacteremia and 69 did not. NE-SFL and NE-WY were significantly higher in patients with bacteremia than in those without bacteremia ( p  < 0.005, respectively), and were significantly correlated with the bacterial load determined by PCR ( r  = 0.384 and r  = 0.374, p  < 0.005, respectively). To assess the diagnostic value for bacteremia, receiver operating characteristic curve analysis was used. NE-SFL and NE-WY showed an area under the curve of 0.685 and 0.708, respectively, while those of PCT, IL-6, presepsin, and CRP were 0.744, 0.778, 0.685, and 0.528, respectively. Correlation analysis showed that the levels of NE-WY and NE-SFL were strongly correlated with PCT and IL-6 levels., Conclusion: This study demonstrated that NE-WY and NE-SFL could predict bacteremia in a manner that may be different from that of other indicators. These findings suggest there are potential benefits of NE-WY/NE-SFL in predicting severe bacterial infections., Competing Interests: NK and YI were employed by the company Sysmex Corporation. Author MK was employed by the company Subsidiary of Sysmex Corporation. The authors declare that this study received funding from Sysmex Corporation. The funder had the following involvement in the study: study design, analysis, interpretation of data. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Miyajima, Niimi, Ueno, Matsui, Higashi, Kojima, Kono, Iwasaki, Nagaoka, Yamamoto and Kitajima.)

Objective To determine the differences between anti-aminoacyl tRNA synthetase (ARS) antibodies among line blots, enzyme-linked immunosorbent assay (ELISA) anti-ARS tests, and RNA-immunoprecipitation (IP) assays. Methods Sera from patients with confirmed or suspected antisynthetase syndrome (ASS) that were positive for either the anti-ARS test or the line-blot assay were used to perform an RNA-IP assay and ELISA to detect individual anti-ARS antibodies. Results Among the 44 patients, 10 were positive only in line-blot assays, 6 were positive only in the anti-ARS test, and 28 were positive in both assays. We compared the accuracy of these assays against the gold standard RNA-IP assay. The κ coefficient was 0.23 in the line-blot assay, but this increased to 0.75 when the cut-off was increased from 1+ to 2+. The κ coefficient was 0.73 in the anti-ARS test. The κ coefficient was 0.85 for positivity in both assays. Patients with ASS that was positive in an RNA-IP assay more frequently had mechanic's hand (62.1% vs. 20%: p=0.031), myositis (51.7 vs. 10%: p=0.028) and more ASS symptoms than those who were positive only in line-blot assays (3.48 vs. 2.2: p=0.019). Conclusions Clinicians need to understand the features of each assay and determine diagnoses by also considering clinical presentations. Diagnoses should not be judged based only on the results of line-blot assays due to the risk of a misdiagnosis from false positives.

Background: Extracellular adenosine 5'-triphosphate (ATP) has been suggested to cause neuroinflammation and motor neuron degeneration by activating microglia and astrocytes in amyotrophic lateral sclerosis (ALS). Since we have developed a highly sensitive ATP assay system, we examined cerebrospinal fluid (CSF) ATP levels in patients with ALS whether it can be a useful biomarker in ALS., Methods: Forty-eight CSF samples from 44 patients with ALS were assayed for ATP with a newly established, highly sensitive assay system using luciferase luminous reaction. CSF samples from patients with idiopathic normal pressure hydrocephalus (iNPH) were assayed as a control. Patients were divided into two groups depending on their disease severity, as evaluated using the Medical Research Council (MRC) sum score. Correlations between the CSF ATP levels and other factors, including clinical data and serum creatinine levels, were evaluated., Results: CSF ATP levels were significantly higher in patients with ALS than in the iNPH (716 ± 411 vs. 3635 ± 5465 pmol/L, p < 0.01). CSF ATP levels were significantly higher in the more severe group than in the iNPH group (6860 ± 8312 vs. 716 ± 411 pmol/L, p < 0.05) and mild group (6860 ± 8312 vs. 2676 ± 3959 pmol/L, p < 0.05) respectively. ALS functional rating scale-revised (ALSFRS-R) (37.9 ± 5.7 vs. 42.4 ± 2.8, p < 0.01) and serum creatinine levels (0.51 ± 0.13 vs. 0.68 ± 0.23 mg/dL, p < 0.05) were significantly lower in the severe group than in the mild group respectively. A negative correlation of CSF ATP levels with MRC sum score was demonstrated in the correlation analysis adjusted for age and sex (r = -0.3, p = 0.08)., Conclusions: Extracellular ATP is particularly increased in the CSF of patients with advanced ALS. CSF ATP levels may be a useful biomarker for evaluating disease severity in patients with ALS.

Background: Several studies have investigated the potential effects of antihistamines on febrile seizure. However, these findings are inconsistent across the studies., Method: A retrospective observational study was conducted on a total of 434 consecutive patients aged between 6 months and 5 years with the diagnosis of febrile seizure. Patients with chronic medical conditions were excluded. Multivariable generalized linear models were conducted to ascertain the effects of antihistamine use on duration of febrile seizure. Also, we conducted a systematic review and meta-analyses of the medical literatures to calculate the pooled estimates using random effects models., Results: The adjusted mean duration of febrile seizure in the antihistamine group was 4.9 min shorter than that in the non-user group (95% confidence interval (CI), 0.4-9.5). The risk of duration in febrile seizure >5 min among antihistamine users was also 0.83 times that among the non-users (95%CI, 0.58-1.19), whereas the risk of duration in febrile seizure >10 min among first-generation antihistamine users was 1.21 times that among non-users (95%CI, 0.69-2.13). According to the systematic review of the literature, 8 observational studies were included in the meta-analyses. Comparing to non-users, the antihistamine users had prolonged duration of febrile seizure by 1.07 min (95%CI, -1.13 to 3.27), elevated risk of duration in febrile seizure >5 min (Risk ratio, 1.16; 95%CI, 0.90-1.49), and similar duration from fever to febrile seizure onset (pooled mean difference, -0.01 h; -1.43 to 1.41), but these estimates were imprecise. Similar results were obtained when we stratified the data by types of antihistamine (first vs. second generation)., Conclusions: Our study may indicate the effects of antihistamine on prolonging febrile seizure duration, but they are still controversial given the limited evidence, highly heterogeneous results, and concerns of the internal and external validities., (Copyright © 2019 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is caused by defective oxidative phosphorylation in the cerebral parenchyma, cerebral blood vessels, and leptomeningeal tissue. Although increased blood and cerebrospinal fluid (CSF) lactate level has been used as a diagnostic biomarker in patients with MELAS, no biomarkers reflecting disease activity exist. Since we have developed a highly sensitive ATP assay system using luciferase luminous reaction, we examined CSF ATP in patients with MELAS and found that it negatively correlates with disease activity and that it reflects the efficacy of the treatment. CSF ATP might be a novel disease monitoring marker for MELAS., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Background: Several cases of hypoglycemia potentially induced by pivalate-conjugated antibiotics have been reported. However, no observational studies have investigated the associations among children., Method: A retrospective cohort study was conducted on 814 consecutive inpatients < 15 years of age with lower respiratory infections. We investigated whether the duration of lower respiratory symptoms and antibiotic use on blood glucose levels and their mediating/moderating effects using multivariable linear regression models and causal mediation analyses. Additionally, we performed a systematic review of the literature that reported the potential associations between pivalate-conjugated antibiotics and hypoglycemia., Results: Multivariable linear regression models showed that the duration of respiratory symptoms and fever had independent relationships with the reduction in blood glucose levels, whereas duration of pivalate-conjugated antibiotic use did not. Causal mediation analyses found that the controlled direct effects of respiratory symptom duration contributed to the reduction in blood glucose levels, but the mediating/moderating effects through antibiotic use did not. A systematic review of the literature included 7 reports written in English and 14 reports written in Japanese. No reports were observational studies; therefore, we were unable to conduct a meta-analysis., Conclusions: Our study failed to demonstrate an association between duration of pivalate-conjugated antibiotic use and blood glucose levels. Further studies are required to illuminate the relationship.

Administering appropriate antimicrobial therapy as early as possible is important for rescuing bacteremic patients. Therefore, rapid antimicrobial susceptibility tests in positive blood culture specimens have been diligently sought. Adenosine triphosphate (ATP) bioluminescence-based methods have been used for rapid antimicrobial susceptibility tests. However, blood culture specimens have not been examined in many studies, possibly due to abundant intracellular ATP in blood corpuscles resulting in false-susceptible results. In this study, we developed a rapid ATP bioluminescence-based method for detecting antibiotic resistance starting from positive blood culture. To minimize background ATP originating from blood corpuscles, specimens were centrifuged and the supernatant diluted with broth, and an ATP-eliminating reagent was then added to the bacterial suspension at the beginning of incubation. This newly devised procedure reduced the background ATP by more than five orders of magnitude. In a pilot study using levofloxacin, no false-susceptible results were observed in 15 clinical specimens. Furthermore, the results indicated that the rapid method provided additional information about bacterial activities with high resolution, in contrast to the less-thorough findings with the conventional turbidity method. Therefore, our approach will contribute to the treatment of infectious diseases as a rapid antimicrobial susceptibility test.

Background: Enarodustat (JTZ-951) is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that mimics adaptive responses to hypoxic conditions and may provide a new therapeutic approach for managing anemia in patients with chronic kidney disease (CKD). We evaluated the efficacy, safety, and maintenance dose of enarodustat in anemic patients with CKD not on dialysis., Methods: Erythropoiesis-stimulating agent (ESA) naïve patients (correction group) and patients on a stable dose of ESA (conversion group) were randomized to receive 2, 4, or 6 mg of enarodustat or placebo once daily for 6 weeks in a double-blind manner (Period 1) followed by 24 weeks of open enarodustat treatment to maintain their hemoglobin (Hb) levels within a target range of 10.0-12.0 g/dL in reference to a dose adjustment algorithm (Period 2)., Results: In the correction group, Hb level increase rate per week increased in a dose-response manner. The proportion of subjects in the conversion group who maintained Hb levels within ± 1.0 g/dL of baseline did not differ between each enarodustat arm and placebo arm during Period 1. Over 70% of subjects in both groups maintained Hb levels within the target range at the end of treatment in Period 2. The mean prescribed doses were 3.58 and 3.74 mg/day in the correction group and the conversion group, respectively. Enarodustat was associated with decreases in hepcidin and ferritin and increased total iron-binding capacity and was generally well tolerated., Conclusions: Enarodustat corrects and maintains Hb levels in anemic patients with CKD not on dialysis., (© 2019 The Author(s) Published by S. Karger AG, Basel.)

Fusion genes involving MEF2D have recently been identified in precursor B-cell acute lymphoblastic leukemia, mutually exclusive of the common risk stratifying genetic abnormalities, although their true incidence and associated clinical characteristics remain unknown. We identified 16 cases of acute lymphoblastic leukemia and 1 of lymphoma harboring MEF2D fusions, including MEF2D-BCL9 (n=10), MEF2D-HNRNPUL1 (n=6), and one novel MEF2D-HNRNPH1 fusion. The incidence of MEF2D fusions overall was 2.4% among consecutive precursor B-cell acute lymphoblastic leukemia patients enrolled onto a single clinical trial. They frequently showed a cytoplasmic μ chain-positive pre-B immunophenotype, and often expressed an aberrant CD5 antigen. Besides up- and down-regulation of HDAC9 and MEF2C , elevated GATA3 expression was also a characteristic feature of MEF2D fusion-positive patients. Mutations of PHF6 , recurrent in T-cell acute lymphoblastic leukemia, also showed an unexpectedly high frequency (50%) in these patients. MEF2D fusion-positive patients were older (median age 9 years) with elevated WBC counts (median: 27,300/ml) at presentation and, as a result, were mostly classified as NCI high risk. Although they responded well to steroid treatment, MEF2D fusion-positive patients showed a significantly worse outcome, with 53.3% relapse and subsequent death. Stem cell transplantation was ineffective as salvage therapy. Interestingly, relapse was frequently associated with the presence of CDKN2A/CDKN2B gene deletions. Our observations indicate that MEF2D fusions comprise a distinct subgroup of precursor B-cell acute lymphoblastic leukemia with a characteristic immunophenotype and gene expression signature, associated with distinct clinical features., (Copyright© 2019 Ferrata Storti Foundation.)

A number of loci are associated with highly heritable schizophrenia and the prevalence of this mental illness has had considerable negative fitness effects on human populations. Here we focused on one particular schizophrenia-associated gene that encodes a sialyltransferase (ST8SIA2) and is expressed preferentially in the brain with the level being largely determined by three SNPs in the promoter region. It is suggested that the expression level of the ST8SIA2 gene is a genetic determinant of schizophrenia risk, and we found that a geographically differentiated non-risk SNP type (CGC-type) has significantly reduced promoter activity. A newly developed method for detecting ongoing positive selection was applied to the ST8SIA2 genomic region with the identification of an unambiguous sweep signal in a rather restricted region of 18 kb length surrounding the promoter. We also found that while the CGC-type emerged in anatomically modern humans in Africa over 100 thousand years ago, it has increased its frequency in Asia only during the past 20-30 thousand years. These findings support that the positive selection is driven by psychosocial stress due to changing social environments since around the last glacial maximum, and raise a possibility that schizophrenia extensively emerged during the Upper Paleolithic and Neolithic era., Competing Interests: The authors have declared that no competing interests exist.

Primates have traditionally been regarded as vision-oriented animals with low olfactory ability, though this "microsmatic primates" view has been challenged recently. To clarify when and how degeneration of the olfactory system occurred and to specify the relevant factors during primate evolution, we here examined the olfactory receptor (OR) genes from 24 phylogenetically and ecologically diverse primate species. The results revealed that strepsirrhines with curved noses had functional OR gene repertoires that were nearly twice as large as those for haplorhines with simple noses. Neither activity pattern (nocturnal/diurnal) nor color vision system showed significant correlation with the number of functional OR genes while phylogeny and nose structure (haplorhine/strepsirrhine) are statistically controlled, but extent of folivory did. We traced the evolutionary fates of individual OR genes by identifying orthologous gene groups, demonstrating that the rates of OR gene losses were accelerated at the ancestral branch of haplorhines, which coincided with the acquisition of acute vision. The highest rate of OR gene loss was observed at the ancestral branch of leaf-eating colobines; this reduction is possibly linked with the dietary transition from frugivory to folivory because odor information is essential for fruit foraging but less so for leaf foraging. Intriguingly, we found accelerations of OR gene losses in an external branch to every hominoid species examined. These findings suggest that the current OR gene repertoire in each species has been shaped by a complex interplay of phylogeny, anatomy, and habitat; therefore, multiple factors may contribute to the olfactory degeneration in primates.

Bitter taste receptors (TAS2R proteins) allow mammals to detect and avoid ingestion of toxins in food. Thus, TAS2Rs play an important role in food choice and are subject to complex natural selection pressures. In our previous study, we examined nucleotide variation in TAS2R38, a gene expressing bitter taste receptor for phenylthiocarbamide (PTC), in 333 Japanese macaques (Macaca fuscata) from 9 local populations in Japan. We identified a PTC "non-taster" TAS2R38 allele in Japanese macaques that was caused by a loss of the start codon. This PTC non-taster allele was only found in a limited local population (the Kii area), at a frequency of 29%. In this study, we confirmed that this allele was present in only the Kii population by analyzing an additional 264 individuals from eight new populations. Using cellular and behavioral experiments, we found that this allele lost its receptor function for perceiving PTC. The nucleotide sequences of the allele including flanking regions (of about 10 kb) from 23 chromosomes were identical, suggesting that a non-taster allele arose and expanded in the Kii population during the last 13,000 years. Genetic analyses of non-coding regions in Kii individuals and neighboring populations indicated that the high allele frequency in the Kii population could not be explained by demographic history, suggesting that positive selection resulted in a rapid increase in PTC non-tasters in the Kii population. The loss-of-function that occurred at the TAS2R38 locus presumably provided a fitness advantage to Japanese macaques in the Kii population. Because TAS2R38 ligands are often found in plants, this functional change in fitness is perhaps related to feeding habit specificity. These findings should provide valuable insights for elucidating adaptive evolutionary changes with respect to various environments in wild mammals.

We discovered a lethal hemorrhagic syndrome arising from severe thrombocytopenia in Japanese macaques kept at the Primate Research Institute, Kyoto University. Extensive investigation identified that simian retrovirus type 4 (SRV-4) was the causative agent of the disease. SRV-4 had previously been isolated only from cynomolgus macaques in which it is usually asymptomatic. We consider that the SRV-4 crossed the so-called species barrier between cynomolgus and Japanese macaques, leading to extremely severe acute symptoms in the latter. Infectious agents that cross the species barrier occasionally amplify in virulence, which is not observed in the original hosts. In such cases, the new hosts are usually distantly related to the original hosts. However, Japanese macaques are closely related to cynomolgus macaques, and can even hybridize when given the opportunity. This lethal outbreak of a novel pathogen in Japanese macaques highlights the need to modify our expectations about virulence with regards crossing species barriers.

Olfactory receptors (ORs) detect odors in the environment, and OR genes constitute the largest multigene family in mammals. Numbers of OR genes vary greatly among species--reflecting the respective species' lifestyles--and this variation is caused by frequent gene gains and losses during evolution. However, whether the extent of gene gains/losses varies among individual gene lineages and what might generate such variation is unknown. To answer these questions, we used a newly developed phylogeny-based method to classify >10,000 intact OR genes from 13 placental mammal species into 781 orthologous gene groups (OGGs); we then compared the OGGs. Interestingly, African elephants had a surprisingly large repertoire (∼ 2000) of functional OR genes encoded in enlarged gene clusters. Additionally, OR gene lineages that experienced more gene duplication had weaker purifying selection, and Class II OR genes have evolved more dynamically than those in Class I. Some OGGs were highly expanded in a lineage-specific manner, while only three OGGs showed complete one-to-one orthology among the 13 species without any gene gains/losses. These three OGGs also exhibited highly conserved amino acid sequences; therefore, ORs in these OGGs may have physiologically important functions common to every placental mammal. This study provides a basis for inferring OR functions from evolutionary trajectory., (© 2014 Niimura et al.; Published by Cold Spring Harbor Laboratory Press.)

Genome studies of mammals in the superorder Euarchontoglires (a clade that comprises the orders Primates, Dermoptera, Scandentia, Rodentia, and Lagomorpha) are important for understanding the biological features of humans, particularly studies of medical model animals such as macaques and mice. Furthermore, the dynamic ecoevolutionary signatures of Euarchontoglires genomes may be discovered because many species in this clade are characterized by their successful adaptive radiation to various ecological niches. In this study, we investigated the evolutionary trajectory of bitter taste receptor genes (TAS2Rs) in 28 Euarchontoglires species based on homology searches of 39 whole-genome assemblies. The Euarchontoglires species possessed variable numbers of intact TAS2Rs, which ranged from 16 to 40, and their last common ancestor had at least 26 intact TAS2Rs. The gene tree showed that there have been at least seven lineage-specific events involving massive gene duplications. Gene duplications were particularly evident in the ancestral branches of anthropoids (the anthropoid cluster), which may have promoted the adaptive evolution of anthropoid characteristics, such as a trade-off between olfaction and other senses and the development of herbivorous characteristics. Subsequent whole-gene deletions of anthropoid cluster TAS2Rs in hominoid species suggest ongoing ectopic homologous recombination in the anthropoid cluster. These findings provide insights into the roles of adaptive sensory evolution in various ecological niches and important clues related to the molecular mechanisms that underlie taste diversity in Euarchontoglires mammalian species, including humans., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Parasites of the genus Plasmodium infect all classes of amniotes (mammals, birds and reptiles) and display host specificity in their infections. It is therefore generally believed that Plasmodium parasites co-evolved intimately with their hosts. Here, we report that based on an evolutionary analysis using 22 genes in the nuclear genome, extant lineages of Plasmodium parasites originated roughly in the Oligocene epoch after the emergence of their hosts. This timing on the age of the common ancestor of extant Plasmodium parasites suggest the importance of host switches and lends support to the evolutionary scenario of a "malaria big bang" that was proposed based on the evolutionary analysis using the mitochondrial genome., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: A multicenter open trial was performed to clarify the optimal duration of combined pegylated interferon (Peg-IFN) plus ribavirin therapy in patients with chronic hepatitis caused by HCV genotype 2., Methods: A total of 100 patients seen between 2005 and 2007 received the combination therapy for 4 to 52 weeks. The cutoff value of the HCV-RNA-negative (titers under 1.7 Log IU/mL) period during the therapy to predict sustained virological response (SVR) was determined by ROC curve and multivariate logistic regression analyses. The result was validated in 48 patients between 2008 and 2009., Results: SVR was achieved in 78 patients. Serum HCV-RNA titers decreased to less than 1.7 Log IU/mL at 4 weeks of the therapy in 60 patients. The SVR rate in these patients was 85%, which was significantly higher than that of remaining 40 patients with a SVR rate of 68%. An HCV-RNA-negative period of ≥17 weeks was selected as the cutoff value, which showed a significant odds ratio of 4.77 for SVR. Among the 35 patients who showed a decrease of the serum HCV-RNA of less than 1.7 Log IU/mL between 8 and 16 weeks of therapy, the SVR rate was significantly higher in 16 patients with a serum HCV-RNA-negative period of ≥17 weeks (94%) than in 19 patients in whom the period was less than 17 weeks (63%). Similar results were obtained in the subsequent validation study., Conclusion: Prolonged combined Peg-IFN plus ribavirin therapy, with an HCV-RNA-negative period of ≥17 weeks, yielded good therapeutic outcomes in patients with chronic HCV genotype 2 hepatitis.

The cellular energy produced by mitochondria is a fundamental currency of life. However, the extent to which mitochondrial (mt) performance (power and endurance) is adapted to habitats and life strategies of vertebrates is not well understood. A global analysis of mt genomes revealed that hydrophobicity (HYD) of mt membrane proteins (MMPs) is much lower in terrestrial vertebrates than in fishes and shows a strong negative correlation with serine/threonine composition (STC). Here, we present evidence that this systematic feature of MMPs was crucial for the evolution of large terrestrial vertebrates with high aerobic capacity. An Arrhenius-type equation gave positive correlations between STC and maximum life span (MLS) in terrestrial vertebrates (with a few exceptions relating to the lifestyle of small animals with a high resting metabolic rate [RMR]) and negative correlations in secondary marine vertebrates, such as cetaceans and alligators (which returned from land to water, utilizing buoyancy with increased body size). In particular, marked STC increases in primates (especially hominoids) among placentals were associated with very high MLS values. We connected these STC increases in MMPs with greater stability of respiratory complexes by estimating the degradation of the Arrhenius plot given by accelerating mtRMR up to mt maximum metabolic rate. Both mtRMR and HYD in terrestrial vertebrates decreased with increasing body mass. Decreases in mtRMR raise MMP stability when high mobility is not required, whereas decreased HYD may weaken this stability under the hydrophobic environment of lipid bilayer. High maximal metabolic rates (5-10 RMR), which we postulate require high MMP mobility, presumably render MMPs more unstable. A marked rise in STC may therefore be essential to stabilize MMPs, perhaps as dynamic supercomplexes, via hydrogen bonds associated with serine/threonine motifs.

Bitter taste perception evolved as a key detection mechanism against the ingestion of bioactive substances, and is mediated by TAS2R gene family members in vertebrates. The most widely known and best studied bitter substance is phenylthiocarbamide (PTC), which is recognized by TAS2R38 and has a molecular structure similar to that of glucosinolates contained in Brassica plants. The "non-taster" phenotypic polymorphism (i.e., not sensitive to PTC-containing foods) has been identified in many primates, including humans. Here, we report genetic and behavioral evidence for the existence of "non-taster" Japanese macaques, which originated from a restricted region of Japan. Comparison of the sequences of the TAS2R38 gene of 333 Japanese and 55 rhesus macaques suggested that this genotype appeared after the divergence of these two species, independently of the appearance of human and chimpanzee "non-tasters". This finding might give a clue for elucidating the ecological, evolutionary, and neurobiological aspects of bitter taste perception of primates, as related to the plants that they sometimes use as foods in their habitats.

Odor molecules in the environment are detected by olfactory receptors (ORs), being encoded by a large multigene family in mammalian genomes. It is generally thought that primates are vision oriented and dependent weakly on olfaction. Previous studies suggested that Old World monkeys (OWMs) and hominoids lost many functional OR genes after the divergence from New World monkeys (NWMs) due to the acquisition of well-developed trichromatic vision. To examine this hypothesis, here we analyzed OR gene repertoires of five primate species including NWMs, OWMs, and hominoids for which high-coverage genome sequences are available, together with two prosimians and tree shrews with low-coverage genomes. The results showed no significant differences in the number of functional OR genes between NWMs (marmosets) and OWMs/hominoids. Two independent analyses, identification of orthologous genes among the five primates and estimation of the numbers of ancestral genes by the reconciled tree method, did not support a sudden loss of OR genes at the branch of the OWMs/hominoids ancestor but suggested a gradual loss in every lineage. Moreover, we found that humans retain larger numbers of ancestral OR genes that were in the common ancestor of NWMs/OWMs/hominoids than orangutans and macaques and that the OR gene repertoire in humans is more similar to that of marmosets than those of orangutans and macaques. These results suggest that the degeneration of OR genes in primates cannot simply be explained by the acquisition of trichromatic vision, and our sense of smell may not be inferior to other primate species.

The streaked tenrec (Hemicentetes semispinosus) is equipped with a quill vibrating mechanism on the dorsal side of the caudal trunk that has evolved as an extraordinary sounding apparatus for communication. An arrangement of 15 or 16 light-brown quills was observed. Thickened cutaneous muscles were confirmed beneath quills. We named this structure the "quill vibrator disc" (QVD). The QVD was 16.8 mm long and 8.55 mm wide in a typical adult. Longitudinal musculature symmetrical about the sagittal plane was developed in the QVD. Myocytes were found immunohistochemically to contain mainly fast myosin but not slow myosin. These findings indicate that the QVD is a specialized apparatus in the cutaneous muscle that contributes to the vibration of quills and to the production of sound for communication.

Prosimians (tarsiers and strepsirrhini) represent the basal lineages in primates and have a close bearing on the origin of primates. Although major lineages among anthropoidea (humans, apes and monkeys) are well represented by complete mitochondrial DNA (mtDNA) sequence data, only one complete mtDNA sequence from a representative of each of the infraorders in prosimians has been described until quite recently, and therefore we newly determined complete mtDNA sequences from 5 lemurs, 4 lorises, one tarsier and one platyrrhini. These sequences were provided to phylogenetic analyses in combination with the sequences from the 15 primates species reported to the database. The position of tarsiers among primates could not be resolved by the maximum likelihood (ML) and neighbor-joining (NJ) analyses with several data sets. As to the position of tarsiers, any of the three alternative topologies (monophyly of haplorhini, monophyly of prosimians, and tarsiers being basal in primates) was not rejected at the significance level of 5%, neither at the nucleotide nor at the amino acid level. In addition, the significant variations of C and T compositions were observed across primates species. Furthermore, we used AGY data sets for phylogenetic analyses in order to remove the effect of different C/T composition bias across species. The analyses of AGY data sets provided a medium support for the monophyly of haplorhini, which might have been screened by the variation in base composition of mtDNA across species. To estimates the speciation dates within primates, we analyzed the amino acid sequences of mt-proteins with a Bayesian method of Thorne and Kishino. Divergence dates were estimated as follows for the crown groups: about 35.4 million years ago (mya) for lorisiformes, 55.3 mya for lemuriformes, 64.5 mya for strepsirrhini, 70.1 mya for haplorhini and 76.0 mya for primates. Furthermore, we reexamined the biogeographic scenarios which have been proposed for the origin of strepsirrhini (lemuriformes and lorisiformes) and for the dispersal of the lemuriformes and lorisiformes.

An excess of nonsynonymous substitutions over synonymous ones has been regarded as an important indicator of adaptive evolution or positive selection at the molecular level. We now report such a case for rbcL sequences among cryptic species in Conocephalum (Hepaticae, Bryophytes). This finding can be regarded as evidence of adaptive evolution in several cryptic species (especially in F and JN types) within the genus. Bryophytes are small land plants with simple morphology. We can therefore expect the existence of several biologically distinct units or cryptic species within each morphological species. In our previous study, we found three rbcL types in Asian Conocephalum japonicum (Thunb.) Grolle and also found evidence strongly suggesting that the three types are reproductively isolated cryptic species. Additionally, we examined rbcL sequence variation in six cryptic species of C. conicum (L.) Dumort. previously recognized by allozyme analyses. As a result, we were able to discriminate the six cryptic species based only on their rbcL sequences. We were able to show that rbcL sequence variation is also useful in finding cryptic species of C. conicum.

Aim: The indications for liver transplantation in cases of fulminant hepatitis are currently determined according to the Guideline of the Acute Liver Failure Study Group of Japan in 1996, which is based on assessment of the prognosis of the patients at the onset of hepatic encephalopathy and reassessed 5 days later. This Guideline was prepared based on the clinical findings in patients seen between 1988 and 1992, and showed a predictive accuracy of 82% in the patients seen between 1993 and 1995. In this study, the usefulness of the Guideline was re-evaluated, since the therapeutic strategies for fulminant hepatitis have advanced remarkably over the last 10 years., Methods: The predictive accuracy of the Guideline was assessed in 698 patients with fulminant hepatitis and late onset hepatic failure (LOHF) between 1998 and 2003. The time-point in the course of the disease at which physicians considered liver transplantation was examined., Results: The accuracy in patients not receiving liver transplantation was 68% and 78% in acute and subacute types, respectively, of fulminant hepatitis, and 84% among LOHF cases. The values did not improve following the reassessment. The sensitivity and specificity of the assessment in patients with acute and subacute types, respectively, were extremely low. Liver transplantation was considered in 42% of LOHF patients at 8 or more days before encephalopathy development., Conclusion: The Guideline should be modified to improve its accuracy. The Guideline should also be made adoptable for the assessment of LOHF patients before hepatic encephalopathy onset.

Aim: A nationwide survey was performed to clarify the present state of fulminant hepatitis and late onset hepatic failure (LOHF) between 1998 and 2003 in Japan., Methods: Three hundred and sixteen, 318 and 64 patients, respectively, with acute and subacute types of fulminant hepatitis and LOHF, in which grade II or more severe hepatic encephalopathy occurred within 10 days, between 11 days and 8 weeks and between 8 and 24 weeks, respectively, after the onset of disease symptoms, were analyzed., Results: Complications such as metabolic syndrome were underlying in 41.5% of patients with subacute fulminant hepatitis and 51.6% of patients with LOHF, and most of such patients had received daily medications. The etiology of fulminant hepatitis was viral infection in 71.2% of the acute type and 31.8% in the subacute type. Hepatitis B virus (HBV) infection was found in most of these patients; transient infection prevailed in the acute type; and HBV carrier prevailed in the subacute type. The etiology was unknown in 42.8% and 53.1% of the subacute type and LOHF, respectively. Autoimmune hepatitis and drug allergy-induced liver injury were found in 10.7% and 11.3%, respectively, of the subacute type. Artificial liver support with plasma exchange and/or hemodiafiltration took place in more than 90% of all patients. The survival rates of the patients without liver transplantation were 53.7% in the acute and 24.4% in the subacute type, and 11.5% in LOHF. The prognosis was especially poor in HBV carriers and patients with autoimmune hepatitis. The survival rates of those who underwent liver transplantation were 56.3%, 39.3% and 23.4% in the acute type, subacute type and LOHF, respectively., Conclusion: The etiology and prognosis differed in patients with fulminant hepatitis and LOHF depending on the disease types in Japan, and liver transplantation improved the prognosis of the patients irrespective of the disease type and etiology.

Infection with cagA-positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms.

Although the majority of hepatitis E virus (HEV) infections in Japan are 'domestic' due to the presence of indigenous strains, there are still 'imported' cases as well. Among 83 patients with non-A, non-B and non-C acute liver diseases admitted to Saitama Medical University Hospital, 7 (8.4%) were positive for serum HEV-RNA, of whom 2 had a recent history of traveling to China, one to Xian and another to Shanghai. We determined the full-genome sequences of HEV from these 2 patients (isolate names are JKO-ChiSai98c and JYI-ChiSai01c, genotype 4 in both) for phylogenetic analyses. Initially, when compared only to the 13 full-genome sequences of genotype 4 so far reported, our 2 isolates were thought to be novel strains because they showed a significant genetic difference from the sequences known to date. However, when we included a set of short sequences (150 nt) recently reported from China in the comparison, we found that our 2 isolates represent a subgroup of genotype 4, which seems to be restricted to eastern China. In conclusion, the 2 HEV isolates reported here could serve as full-genome prototypes for an eastern China-indigenous subgroup of the genotype 4 HEV., ((c) 2007 S. Karger AG, Basel.)

A new apoptosis cascade mediated by lysosomal lactoferrin was found in apoptotic liver induced by d-galactosamine. Caspase-3 and lactoferrin were increased in the apoptotic liver cytoplasm and serum transaminases were elevated. Recombinant lactoferrin stimulated procaspase-3 processing at 10(-6)-10(-7)M to an extent similar to that by granzyme B in vitro. Lactoferrin changed procaspase-3 structure susceptible to the processing. Synthetic peptide Y(679)-K(695) in lactoferrin molecule inhibited the processing of procaspase-3 by lactoferrin. Lactoferrin in lysosomes was decreased and lactoferrin released into cytoplasm was increased quantitatively in d-galactosamine induced apoptotic liver, and procaspase-3 in cytoplasm was processed to caspase-3.

The ancestor(s) of apparently Japan-indigenous strains of Hepatitis E virus (HEV) was probably of foreign origin, but it remains unclear when and from where it made inroads. In this study, 24 genotype 3 and 24 genotype 4 HEV strains recovered in Japan each showed a significant cluster, clearly distinct from those of foreign strains, in the phylogenetic tree constructed from an 821 nt RNA polymerase gene fragment. The evolutionary rate, approximately 0.8 x 10(-3) nucleotide substitutions per site per year, enabled tracing of the demographic history of HEV and suggested that the ancestors of Japan-indigenous HEV had made inroads around 1900, when several kinds of Yorkshire pig were imported from the UK to Japan. Interestingly, the evolutionary growth of genotype 3 in Japan has been slow since the 1920s, whereas genotype 4 has spread rapidly since the 1980s. In conclusion, these data suggest that the indigenization and spread of HEV in Japan were associated with the popularization of eating pork.

Osteopontin is a crucial factor for initiation of Th1 immune reaction. Previously, we established transgenic mice expressing osteopontin in hepatocytes, in which lymphocyte infiltration occurred spontaneously at 12 weeks of age and liver necrosis at 24 weeks of age. This liver necrosis may develop through provocation by excessive Th1 immune reaction, but it is also possible that hepatocytes become fragile under abundant osteopontin in the cytoplasm. Thus, gene expression profiles in the liver were evaluated to seek such contributing factors in the transgenic mice. On DNA microarray analysis of 3774 mouse genes, 16 genes were selected as hepatic genes significantly up-regulated in the transgenic mice aged 8 weeks than in the negative littermate, which included mRNAs of cytoprotective metallothionein and glutathione S-transferase (GST). Hepatic up-regulations of both genes were also seen by Western blotting. Liver necrosis in the centrilobular areas developed after carbon tetrachloride treatment, but its histological extent and plasma ALT activities were significantly smaller in the transgenic mice aged 8 weeks than in the wild-type C57BL/6 control mice. We conclude that cytoprotective function of the liver is increased through up-regulated expressions of metallothionein and GST, and thereby susceptibility of hepatocytes to the stress may be less possible, if any, in the development of spontaneous liver necrosis in transgenic mice expressing osteopontin in hepatocytes.

Osteopontin is a cytokine essential for initiation of Th1 immune reaction. We established transgenic mice expressing osteopontin in hepatocyte, in which liver necrosis with lymphocyte infiltration developed gradually from 12 weeks of age with up-regulated osteopontin levels in the circulation, suggesting that extrahepatic manifestations might also occur as a result of excessive Th1 immune reaction. We examined histological and immunohistochemical features of various organs in these mice. Splenomegaly and enlargement of lymph nodes around the liver and intestine became apparent with marked infiltration of small lymphocytes in the transgenic mice later than 24 weeks of age. Immunostaining revealed that lymphocytes in the spleen and lymph nodes were positive for either CD3 or CD20, suggesting that the infiltrating lymphocytes were both B and T cells. Similar lymphocyte infiltration was found in the lung, kidney and submandibular gland. Alveolar septa became hypertrophic with lymphocyte infiltration, and the lung showed the appearance of interstitial pneumonia. These lesions are similar to extrahepatic manifestations in chronic hepatitis C patients, suggesting that augmented Th1 immune reaction to hepatitis C virus (HCV) proteins or the proteins with molecular mimicry of HCV may be a contributing factor for the formation of the pathological state not only in the liver but also in various organs under chronic infection of hepatitis C virus.

Background: The T-helper (Th)1 immune reaction is essential for the eradication of hepatitis C virus (HCV) during interferon (IFN) therapy in patients with chronic hepatitis C. Osteopontin is a cytokine crucial for the initiation of the Th1 response. Recently, we identified four single-nucleotide polymorphisms (SNPs) in the promoter region of the osteopontin gene (OPN), at nucleotide (nt) -155, -443, -616, and -1748, and suggested that the SNP at nt -443 was a marker reflecting hepatitis activity in patients with HCV. Therefore, we examined the possibility that SNPs in OPN were also markers predicting the therapeutic efficacy of IFN in patients with chronic hepatitis C., Methods: Blood was collected from 77 patients with chronic hepatitis C who had received either IFN monotherapy or IFN-ribavirin combination therapy (IFN-based therapies). SNPs in OPN, MxA, MBL, and LMP7 were analyzed by Invader assay., Results: Promoter SNPs of OPN at nt -155, -616, and -1748 showed linkage disequilibrium at 100% to each other. Sustained virological response (SVR) was observed in 58% of all patients. The SVR rate was higher in patients with the G/G or G/A alleles in the OPN promoter SNP at nt -1748 than in those with A/A (85% vs 45%; P < 0.05). The SVR rate was also higher in patients with T/T at nt -443 than in those with C/C or C/T (86% vs 47%; P < 0.05). Such differences were particularly evident in patients with HCV genotype 1b who had a pretreatment viral load greater than 100 KIU/ml. All the patients who had G/G or G/A at nt -1748 and T/T at nt -443 obtained an SVR. On the other hand, there was no relationship between the efficacy of IFN-based therapies and SNPs in MxA, MBL, and LMP7, which had been shown to have association with the response to IFN monotherapies., Conclusions: SNPs in the promoter region of OPN may be useful as a marker to predict the efficacy of IFN-based therapies in patients with chronic hepatitis C, and further investigation regarding their real significance is warranted in a large series of patients.

Background/aims: Inchin-ko-to (TJ-135) is an herbal medicine used in Japan for treatment of icteric patients with cirrhosis. Its efficacy as an anti-fibrogenic drug was evaluated in relation to stellate cell activation., Methods/results: Liver fibrosis was induced in rats by repeated injections of carbon tetrachloride (CCl4) or pig-serum. Oral administration of TJ-135 improved the mortality of rats given CCl4 with reduced extents of liver necrosis and fibrosis. Similar improvement of liver fibrosis was found in rats given pig-serum showing no liver necrosis. DNA synthesis of stellate cells activated in vitro after isolation from normal rat liver was decreased by culture with TJ-135 in a dose-related manner, accompanied by decreased smooth muscle alpha actin expression and contractility. Such attenuation was not found in the cells cultured with geniposide, an iridoid compound of TJ-135, but genipin, an aglycone of geniposide formed in the gut by action of bacterial flora, markedly decreased stellate cell activation without affecting synthesis of proteins other than collagen., Conclusions: TJ-135 may be useful for treatment of liver fibrosis and portal hypertension through suppression of activated hepatic stellate cell function by genipin, an absorbed form of its component.

Background: Massive liver necrosis can develop as a consequence of imbalance between T-helper (Th)1 and Th2 immune reactions in the liver. Osteopontin is a glycoprotein secreted for the initiation of the Th1 immune reaction, as well as for extracellular matrix formation and calcium deposition in the bone and kidney. Osteopontin is overexpressed in Kupffer cells, macrophages, and stellate cells activated in injured livers. We established transgenic mice expressing osteopontin exclusively in hepatocytes, using a vector containing human serum amyloid P component promoter. The relation of Th1/Th2 immune imbalance to massive liver necrosis was studied using these transgenic mice., Methods: Transgenic mice and C27BL/6 mice, wild-type controls of the transgenic mice, were given an intravenous injection of concanavalin-A, and the histological extent of liver injuries and plasma cytokine levels were evaluated., Results: When the transgenic mice received concanavalin-A, massive necrosis and mononuclear cell infiltration developed in the liver, the extent of which was greater in the female mice than in the male mice. This treatment produced minimal liver injury and focal liver necrosis in male and female C57BL/6 mice. In these transgenic and control mice, plasma concentrations of interleukin (IL)-10 and interferon (IFN)-gamma were increased after concanavalin-A treatment. However, the upregulation of plasma IL-10 concentration was smaller in the male and female transgenic mice than in the control mice, and the upregulation of the IFN-gamma concentration was greater in the female transgenic mice than in the female control mice., Conclusions: Th1 and Th2 immune reactions were deranged after concanavalin-A treatment, with Th1 immunity predominating in transgenic mice expressing osteopontin in hepatocytes; this immunological imbalance may contribute to massive liver necrosis.

Fulminant hepatitis is characterized by massive or submassive liver necrosis. Massive liver necrosis can be induced by activated macrophages infiltrating into the liver. Osteopontin, an extracellular matrix, is a secretory glycoprotein as well essential for Th1 immune response, contributing to macrophage activation and infiltration. To know the significance of osteopontin in the development of fulminant hepatitis, plasma osteopontin levels were measured in patients with fulminant hepatitis. The levels were significantly greater in patients with fulminant hepatitis than in those with acute or chronic hepatitis as well as healthy adults. Among patients with fulminant hepatitis except one in whom bacterial infection was complicated, plasma osteopontin levels were elevated especially in the patients who developed hepatic encephalopathy of grade II or more within 10 days of the disease onset, a clinical type characteristic of massive liver necrosis. Immunohistochemical examination revealed that osteopontin was stained in macrophages positive for CD68, a marker for macrophages, in necrotic areas of the liver in a patient with fulminant hepatitis. In conclusion, plasma osteopontin levels were elevated in patients with fulminant hepatitis, probably reflecting production of osteopontin in Kupffer cells and hepatic macrophages, which might be involved in the development of massive liver necrosis in fulminant hepatitis.

Sinusoidal endothelial cells and hepatocytes increase in number after partial resection and necrosis of the liver, and contribute to both reconstruction and capillarization of the sinusoids through interaction with stellate cells. The mechanism underlying such interaction was evaluated regarding angiopoietins and tie receptors essential for blood vessel formation. Hepatic mRNA expressions of angiopoietin-1, angiopoietin-2 and tie-2 were increased at 168h after 70% liver resection with sinusoidal reconstruction in rats, and also at 48h with sinusoidal capillarization in the liver of rats given carbon tetrachloride (CCl(4)). Tie-2 mRNA expression was detected in sinusoidal endothelial cells and stellate cells isolated from normal rats, and in activated stellate cells from CCl(4)-intoxicated rats. The mRNA expressions of angiopoietin-1 and angiopoietin-2 were detectable in Kupffer cells, sinusoidal endothelial cells and stellate cells in normal rats, but increased in activated stellate cells and macrophages after CCl(4)-intoxication. Both angiopoietins and tie-2 were immunohistochemically stained along the sinusoids of 70% hepatectomized rats and in necrotic areas of CCl(4)-intoxicated rats. Angiopoietin-1 and angiopoietin-2 may be involved in both reconstruction and capillarization of the sinusoids in rat liver after partial resection and necrosis through interaction between stellate cells and sinusoidal and vascular endothelial cells via tie-2 receptor.

Osteopontin, a crucial factor for Th1 immune response, is expressed in stellate cells and macrophages activated in injured liver. To clarify the role of osteopontin in inflammatory changes in the liver, we attempted to establish transgenic mice expressing osteopontin in hepatocytes. Mouse osteopontin cDNA, cloned from concanavalin-A-stimulated spleen cells in C57BL/6 mice, was constructed into the vector containing serum amyloid-P component promoter. This construction was microinjected into fertilized eggs of C57BL/6 mice, and 4 lines of the transgenic mice were obtained. Western blotting and immunohistochemistry revealed that osteopontin was expressed in hepatocytes, but not in non-parenchymal cells, in the transgenic mice. The mean osteopontin concentrations in the liver and plasma in the mice were 13 and 2.6 times higher than those in negative littermates. Antinuclear antibody was positive in the plasma in 50% of the transgenic mice. In the transgenic mice later than 12 weeks of age, mononuclear cell infiltration in the liver developed, and these cells were positive for CD8 and HLA-DR. Plasma ALT activity was increased with focal necrosis in hepatic lobules in the transgenic mice later than 24 weeks of age. The transgenic mice expressing osteopontin in hepatocytes may be useful as a model of autoimmune hepatitis.

Background and Aims: Osteopontin, an extracellular matrix protein with RGD motif, is shown to be a cytokine essential for Th1 immune response initiation. Genetic polymorphisms in the osteopontin gene (OPN) determine the magnitude of immunity against rickettsial infection in mice. Similar polymorphisms, if present also in human beings, might affect hepatitis activity in those infected with HCV., Methods: Blood was collected from 176 patients with chronic hepatitis C. SNPs in the promoter region of OPN were analyzed in 20 patients by direct sequencing of DNA fragments amplified by PCR and in 156 patients by Invader assay. Ninety-five patients compatible to evaluation criteria were classified into three groups depending on maximal serum ALT levels during the observation periods at least for 2 years as follows; lower than 30IU/L (low-activity group), between 30 and 80IU/L with no hepatoprotective treatment (medium-activity group), and higher than 80IU/L irrespective of hepatoprotective treatment (high-activity group)., Results: There were 16, 19, and 60 patients in the low-, medium-, and high-activity groups, respectively. Four SNPs (nt -155, -443, -616, and -1748) were detected in the promoter region of OPN. Among them, the SNP at nt -443 (C or T) was a novel one and showed an association with hepatitis activity in our patients: T/T homozygosity was found in 2 (13%), 8 (42%), and 25 (44%), and C/T heterozygosity in 12 (75%), 8 (42%), and 23 (40%), in the low-, medium-, and high-activity groups, respectively. The other 3 SNPs already known showed linkage disequilibrium with D(') and r(2) greater than 0.937 to each other without correlation to disease activity., Conclusions: OPN promoter region SNP at nt -433 may be a useful marker reflecting hepatitis activity in chronic hepatitis C patients.

Vascular endothelial growth factor (VEGF) can induce proliferation of endothelial cells through VEGFR-1 and VEGFR-2 as its receptors, but the intracellular signaling pathway via VEGFR-1 is still unclear. Previously we reported that stellate cells expressed VEGFR-1 exclusively during activation in the liver. In the present paper, the signaling pathway via VEGFR-1 was studied using rat stellate cells activated in vitro. Western blot analysis revealed that both ERK/mitogen-activated protein kinase (MAPK) and PI3K/Akt were phosphorylated in the cells activated by culture in Dulbecco's modified Eagle medium containing 10% fetal calf serum on plastic dishes for 9 days. When VEGF was added to the culture medium, the extent of such PI3K/Akt phosphorylation was increased despite that DNA synthesis and ERK/MAPK phosphorylation were unchanged in the cells. However, this up-regulation of PI3K/Akt phosphorylation was markedly diminished following addition of GFX and PD-98059, inhibitors for protein kinase C (PKC) and ERK/MAPK, respectively. Also, addition of GFX reduced phosphorylation of ERK/MAPK in the cells. It is suggested that VEGF may stimulate signal transduction of PI3K/Akt through VEGFR-1 dependently on activation of the PKC and ERK/MAPK pathway in activated hepatic stellate cells.

Using a rat myofibroblast-like hepatic stellate cell line, we studied the actomyosin-based cytoskeletal actions mediated by Rho-kinase and/or myosin light chain kinase (MLCK). Calmodulin/MLCK inhibitors W-7 and ML-7 attenuated cell migration dose-relatedly at concentrations from 10(-6) to 10(-4)M and collagen gel-contraction by the cells at 10(-4)M, respectively. Rho-kinase inhibitors Y-27632 and HA1077 attenuated the gel-contraction at concentrations from 10(-6) to 10(-4) M, respectively. These Rho-kinase inhibitors attenuated cell migration at 10(-7)M but enhanced the migration at 10(-4)M, respectively. They altered cell morphology showing prominent peripheral actin bundles and sparse central stress fibers, in comparison with the calmodulin/MLCK inhibitors. Both ML-7 and Y-27632 attenuated phosphorylation of myosin regulatory light chain and cell attachment to extracellular substrate. ML-7 attenuated the activation of GTP-binding protein Rac, while Y-27632 did not. These findings suggest that the actomyosin-based cytoskeletal actions can be functionally diverse depending on the Rho-kinase-mediated pathway and the MLCK-mediated pathway.

A highly regio- and stereoselective alpha-methylation reaction of gamma,delta-epoxy-alpha,beta-unsaturated esters was achieved by using a Me2Zn-CuCN reagent.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.

Background: Serum transaminase levels are frequently elevated in patients with chronic hepatitis C who are receiving interferon (IFN)-beta therapy, despite hepatitis C virus (HCV)-RNA being eradicated from the serum. We examined liver histology to determine the reason for this elevation., Methods: Patients with chronic hepatitis C, diagnosed by liver histology and positive serum HCV-RNA, were given intravenous injections of IFN-beta, at a daily dose of 6 MU, every day for periods of 6 to 12 weeks. When serum alanine transaminase (ALT) levels during the therapy were higher than three times the levels before the therapy, liver biopsy was performed. Histological findings on light microscopy were compared in liver biopsy specimens obtained before and during the therapy., Results: An increase in serum ALT levels was found in 19 of the 102 patients who received the IFN-beta therapy. Autoimmune hepatitis was not contributory in any of these 19 patients, because serum antinuclear antigen was negative and IgG levels were not increased. Liver histology was examined in 10 of these 19 patients. The period between the start of IFN-beta therapy and the biopsy during the therapy ranged from 14 to 46 days. In 2 patients, the extent of mononuclear cell infiltration in the liver and hepatocyte necrosis was less than the extent before the therapy. In the remaining 8 patients, the grade of chronic hepatitis was unchanged during,the therapy, but vacuole formation and apoptotic nuclei in hepatocytes were found in 2 patients, and centrilobular necrotic areas in 1 patient., Conclusions: The elevation of serum ALT levels during IFN-beta therapy in chronic hepatitis C patients was not a result of increased hepatitis activity. Degenerative, apoptotic, and necrotic changes in hepatocytes, probably a result of the cytotoxic effects of IFN-beta, may have contributed to this elevation of ALT levels. However, such changes were absent in most of the patients, suggesting that decisions on the discontinuation of IFN-beta therapy must be made in accordance with liver histology findings.

Regulatory mechanisms of expressions of receptors for vascular endothelial growth factor (VEGF) are an important issue to clarify angiogenesis in the liver. Effects of exogenous VEGF on VEGF receptor expressions and intracellular signal transduction system were examined using human umbilical venous endothelial cells (HUVECs). HUVECs were cultured in BEM-2 solution containing VEGF for 5 days, and subjected to experiments 24 h after replacement of the medium to EBM-2 solution without VEGF. Addition of VEGF at concentrations up to 100 ng/ml increased the incorporation of 3H-thymidine into the DNA in the cells in a dose-related manner. Similar increases were found in mRNA expressions of VEGF receptors, VEGFR-1 and VEGFR-2, assessed by Northern blotting and in phosphorylation of mitogen activated protein kinase (MAPK) by Western blotting. Such VEGF-induced upregulation of DNA synthesis and phosporylation of MAPK in the cells were attenuated by the addition of GFX, a specific inhibitor of protein kinase C (PKC), but mRNA expression of VEGFR-2 evaluated by quantitative competitive RT-PCR method was not changed. It is suggested that exogenous VEGF upregulated mRNA expressions of VEGF receptors through intracellular signal transduction independent of the PKC-to-MAPK pathway in HUVECs.

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver., (© 1998 The Official Publication of the Asian Pacific Association for the Study of the Liver and the Asian Pacific Association of Gastroenterology.)