Diversity of MHC class I alleles in Spheniscus humboldti.

The major histocompatibility complex locus (MHC) is a gene region related to immune response and exhibits a remarkably great diversity. We deduced that polymorphisms in MHC genes would help to solve several issues on penguins, including classification, phylogenetic relationship, and conservation. This study aimed to elucidate the structure and diversity of the so far unknown MHC class I gene in a penguin species. The structure of an MHC class I gene from the Humboldt penguin (Spheniscus humboldti) was determined by using an inverse PCR method. We designed PCR primers to directly determine nucleotide sequences of PCR products from the MHC class I gene and to obtain recombinant clones for investigating the diversity of the MHC class I gene in Humboldt penguins. A total of 24 MHC class I allele sequences were obtained from 40 individuals. Polymorphisms were mainly found in exons 2 and 3, as expected from the nature of MHC class I genes in vertebrate species including birds and mammals. Phylogenetic analyses of MHC class I alleles have revealed that the Humboldt penguin is closely related to the Red Knot (Calidris canutus) belonging to Charadriiformes.

Association of sick building syndrome with neuropathy target esterase (NTE) activity in Japanese.

Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP-induced delayed neuropathy (OPIDN) and nerve axon degeneration. The aim of our study was to determine whether there was a difference in NTE activity in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with SBS and healthy controls and whether PNPLA6 (alias NTE) gene polymorphisms were associated with SBS. We found that the enzymatic activity of NTE was significantly higher (P < 0.0005) in SBS patients compared with controls. Moreover, population with an AA genotype of a single nucleotide polymorphism (SNP), rs480208, in intron 21 of the PNPLA6 gene strongly reduced the activity of NTE. Fifty-eight SNP markers within the PNPLA6 gene were tested for association in a case-control study of 188 affected individuals and 401 age-matched controls. Only one SNP, rs480208, was statistically different in genotype distribution (P = 0.005) and allele frequency (P = 0.006) between the cases and controls (uncorrected for testing multiple SNP sites), but these were not significant by multiple corrections. The findings of the association between the enzymatic activity of NTE and SBS in Japanese show for the first time that NTE activity might be involved with SBS.

Senescent case of cholesterol ester storage disease that progressed to liver cirrhosis with a novel mutation (N250H) of lysosomal acid lipase gene.

The patient, a 69-year-old man, had a chief complaint of hepatomegaly. The liver was palpated four fingerbreadths below the costal margin, and the spleen was three fingerbreadths below the costal margin. There were no other abnormal findings. Laparoscopy showed that the liver resembled an orange-yellow crayon in appearance and was nodular. The pathological findings of the liver biopsy tissue were consistent with liver cirrhosis. Inside the fibrous septum was an apparent aggregation of enlarged macrophages that phagocytosed lipid components, as well as enlarged Kupffer cells that phagocytosed lipid droplets. Electron microscopy showed the lipid droplets to have a moth-eaten appearance. Using monocytes extracted from the peripheral blood, acid lipase activity was measured by fluorescence spectrometry using 4-methylumbelliferone palmitate as a substrate. This patient's human lysosomal acid lipase activity was 0.020 nM/min per 10(6) cells, corresponding to 5.9% of that in healthy subjects (0.332 ± 0.066 nM/min per 10(6) cells). Cholesterol ester storage disease was therefore diagnosed. The acid lipase A base sequence obtained from leukocytes by direct sequencing was compared with a library. This patient had a point mutation of N250H/N250H in exon 7, a novel gene abnormality that has not previously been reported.

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

A liver-derived immunosuppressive factor is an arginase: identification and mechanism of immunosuppression.

We found a substance in culture medium of neonatal pig liver fragments, which suppresses an immune response monitored by (3)H-thymidine incorporation using phytohemagglutinin (PHA)-stimulated lymphocytes. We named it as an immunosuppressive factor (ISF). To purify ISF, ammonium sulfate fractionation, DE52, SP-Sephadex, hydroxyapatite, blue Sepharose, heparin Sepharose and Superdex gel filtration columns were used. Using these purification procedures, ISF was purified 1,254-fold, with 9.2% recovery, from the culture medium of neonatal pig liver fragments, and was identified as arginase by its biochemical characteristics including molecular size, amino acid sequences of digested peptides and expression of arginase activity. The addition of ISF caused to decrease in arginine concentration in culture medium and at the same time DNA synthesis was suppressed dose-dependently, both of which were recovered by the addition of NOHA (N(G)-hydroxy-L-arginine), an arginase inhibitor. In addition, the depletion of arginine in culture medium also led to the inhibition of DNA synthesis. These results led us to the conclusion that immunosuppressive effect of ISF was due to arginase activity that decreased arginine concentration in culture medium, not to another function of ISF.

Hepatocyte proliferation factors from neonatal pig liver: purification and characterization.

Two factors were found in the condition medium of neonatal pig liver fragments, which were capable of stimulating DNA synthesis in primary hepatocytes. They were named hepatocyte proliferation factor (HPF)-1 and HPF-2 and purified 1,025- and 2,580-fold, respectively. Both HPF-1 and HPF-2 seem to be anionic at pH 8.0 judged from the elution pattern of DEAE (DE52) column chromatography. HPF-1 was recovered as a non-adsorbed fraction in blue Sepharose and heparin Sepharose columns, and had a molecular weight of 26-31 kDa as estimated by gel filtration in high salt condition. Purified HPF-1 stimulated DNA synthesis of primary rat hepatocytes, but suppressed that of HepG2 cells. HPF-2 strongly bound to blue Sepharose and heparin Sepharose columns, and had a molecular weight of 71-90 kDa as estimated by SDS-PAGE under non-reduced condition. Purified HPF-2 stimulated DNA synthesis of primary rat hepatocytes dose dependently but did not suppress that of HepG2 cells. From further biological and chemical characteristics studied in this paper, HPF-1 and HPF-2 may be novel stimulating proteins for hepatocyte proliferation, although the possibility that they are already known growth factors can not be excluded without complete purification and its cloning.

Effects of intravenous amino acids on anesthesia-induced hypothermia in ovariectomized rats.

A decrease in core temperature during general anesthesia is attenuated by infusion of an intravenous amino acid mixture. The purpose of the present study was to investigate the influence of physical and endocrine changes caused by ovariectomy on the inhibitory effect of amino acid infusion on anesthesia-induced hypothermia. Sprague-Dawley female rats were divided into a sham-operated (Sham) group and an ovariectomized (OVX) group. Saline solution or an amino acid mixture solution was infused for 180 min during sevoflurane anesthesia, and the rectal temperature was measured (4 groups). Intraperitoneal white adipose tissue mass, bilateral gastrocnemius weight and plasma insulin levels were measured. In the Sham rats, no inhibitory effect of the amino acid mixture on anesthesia-induced hypothermia was found, while in the OVX rats, hypothermia was significantly decreased. The intraperitoneal fat weight/body weight ratio was significantly higher in the OVX rats than in the Sham rats, but the gastrocnemius weight/body weight ratio was not significantly different. After amino acid infusion, the plasma insulin level was significantly higher in the OVX rats than in the Sham rats. In conclusion, our findings suggest that, in rats, ovarian function or female hormone affects protein turnover mediated by increase in insulin secretion and, thus, decreases the inhibitory effect of an infusion of amino acid mixture on anesthesia-induced hypothermia.

The effect of amino acid infusion on anesthesia-induced hypothermia in muscle atrophy model rats.

An infusion of amino acids stimulates heat production in skeletal muscle and then attenuates the anesthesia-induced hypothermia. However, in a clinical setting, some patients have atrophic skeletal muscle caused by various factors. The present study was therefore conducted to investigate the effect of amino acids on the anesthesia-induced hypothermia in the state of muscle atrophy. As the muscle atrophy model, Sprague-Dawley rats were subjected to hindlimb immobilization for 2 wk. Normal rats and atrophy model rats were randomly assigned to one of the two treatment groups: saline or amino acids (n=8 for each group). Test solutions were administered intravenously to the rats under sevoflurane anesthesia for 180 min, and the rectal temperature was measured. Plasma samples were collected for measurement of insulin, blood glucose, and free amino acids. The rectal temperature was significantly higher in the normal-amino acid group than in the muscle atrophy-amino acid group from 75 to 180 min. The plasma insulin level was significantly higher in the rats given amino acids than in the rats given saline in both normal and model groups. In the rats given amino acids, plasma total free amino acid concentration was higher in the model group than in the normal group. These results indicate that skeletal muscle plays an important role in changes in body temperature during anesthesia and the effect of amino acids on anesthesia-induced hypothermia decreases in the muscle atrophy state. In addition, intravenous amino acids administration during anesthesia induces an increase in the plasma insulin level.

A novel in vitro system for studying cardiomyocyte differentiation with medaka embryonic cells.

Our studies revealed that dissociated cells from medaka (the fresh-water fish, Oryzias latipes ) blastula-stage embryos differentiate into many rhythmically contracting cells when incubated with a conditioned medium from a cell line. Analyses of these cells by immunostaining, electron microscopy, expression of marker genes, action potential recordings and pharmacological responses all showed the characteristics of myocardial cells. We succeeded in purifying the cardiac cell-inducing factor from the conditioned medium by 523,100-fold with 8% recovery of the protein. Analysis of its amino-acid sequence by mass spectrometry revealed the factor to be medaka activin A2 (homodimer of inhibin betaA2 subunit). Recombinant human activin A showed the same cardiac cell-inducing activity toward medaka embryonic cells. Our results demonstrate that activin A is a potent factor for medaka myocardial cell differentiation. This culture method should provide a novel and simple experimental system to study cardiomyocyte differentiation in vitro.

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti).

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.

Assessment of a difference in ALDH2 heterozygotes and alcoholic liver injury.

We have speculated that the degree of liver dysfunction in alcoholic liver disease with ALDH2*1/2*2 may be less pronounced than that with ALDH2*1/2*1. In the present study, outpatients with alcoholic liver injury were examined for ALDH2 genotype and biochemical data. The number of patients was 29 cases of nonspecific changes, 16 cases of fatty liver, 5 cases of liver fibrosis, and 44 cases of liver cirrhosis. Biochemical data were evaluated with ALDH2 heterozygotes data obtained by PCR-SSCP. The ALDH2*1/2*1 and ALDH2*1/2*1 genotypes accounted for 90% and 10%, respectively. As for ALDH2*1/2*2, there were three patients with nonspecific changes, three with fatty liver, one with liver fibrosis, and two with liver cirrhosis. In alcoholic liver disease patients, when the ALDH2*1/2*2 genotype was compared with the ALDH2*1/2*1 genotype with biochemical data, the gamma-GTP value in patients with ALDH2*1/2*2 was significantly higher than with ALDH2*1/2*1 ( < 0.005). When the frequency of ALDH2 genotype was determined in patients with alcoholic liver injury, ALDH2 heterozygotes accounted for 15% for the non-cirrhosis group, and 5% for the cirrhotic group. When a relationship between the amount of ethanol intake and biochemical data were determined in patients with alcoholic liver injury who have ALDH2 heterozygotes, the glutamic oxaloacetic transaminase (GOT) and gamma-GTP values were significantly higher at an ethanol intake amount of ethanol more than 100 g per day than intake less than 100 g per day ( < 0.05). The alcoholic patients with ALDH2*1/2*2 drink a slight amount of ethanol, the liver injury is found to be stronger than those with ALDH2*1/2*1 when they drink more than 100 g ethanol per day.