The medaka genome: why we need multiple fish models in vertebrate functional genomics.

Medaka (Oryzias latipes) is widely used in research in the fields of biology, medicine, environmental science and fisheries. Zebrafish and medaka are well established as genetic model systems in which large-scale mutagenesis has been successfully performed, and for which EST data, BAC libraries, and fine linkage maps have been accumulated. Among rayfinned fish, there is a large evolutionary distance between medaka and zebrafish. In contrast, the evolutionary distance between medaka and two species of pufferfish, fugu (Takifugu rubripes), and tetraodon (Tetraodon nigroviridis), is almost comparable to that between humans and rodents, and the current genome project is showing that their genome organization is well conserved. Comparison of genome structure among teleosts and mammals helps our understanding of the orthologous gene structure and the evolution of gene families in vertebrates. In addition, gene functions have to be analyzed by both forward and reverse genetics. The Targeting Induced Local Lesions IN Genome (TILLING) system, which includes random mutagenesis, followed by screening for induced mutations in the target genes, is a powerful tool for studying the functional genomics of both medaka and zebrafish.

Altered expressions of glutamate transporter subtypes in rat model of neonatal cerebral hypoxia-ischemia.

Glutamate transporters are essential for maintaining the extracellular levels of glutamate at synaptic clefts and are regulated developmentally in a subtype-specific manner. We investigated chronological changes of immunoreactivities for glial glutamate transporters GLAST and GLT-1 and a neuronal glutamate transporter, EAAC1, in postnatal 7-day-old rat neocortices and hippocampi at 12, 24, 48 and 72 h after hypoxia-ischemia. Glutamate transporter subtypes are differentially expressed in the ischemic core and the boundary area of the neonatal rat brain with hypoxia-ischemia. Expressions of these glutamate transporters decreased in the ischemic core at 12 h, then immunoreactivities for GLAST and GLT-1 were recovered at the hippocampus. This was accompanied by a GFAP-positive gliosis at 72 h, whereas these immunoreactivities were reduced at the neocortex in the ischemic core. Glial glutamate transporters, especially GLAST, were noted in some astrocytes appearing as apoptosis as well as shrunken pyramidal neurons mainly in the boundary area of the neocortex. Increased perikaryal expression of EAAC1 was associated with that of MAP2 at the border of the boundary area. These temporal and regional expressions of glutamate transporters may contribute towards understanding the excitotoxic cell death mechanism in hypoxic-ischemic encephalopathy during the perinatal period.

Mutations in the gene encoding B, a novel transporter protein, reduce melanin content in medaka.

Pigmentation of the skin is of great social, clinical and cosmetic significance. Several genes that, when mutated, give rise to altered coat color in mice have been identified; their analysis has provided some insight into melanogenesis and human pigmentation diseases. Such analyses do not, however, fully inform on the pigmentation of lower vertebrates because mammals have only one kind of chromatophore, the melanocyte. In contrast, the medaka (a small, freshwater teleost) is a suitable model of the lower vertebrates because it has all kinds of chromatophores. The basic molecular genetics of fish are known and approximately 70 spontaneous pigmentation mutants have been isolated. One of these, an orange-red variant, is a homozygote of a well-known and common allele, b, and has been bred for hundreds of years by the Japanese. Here, we report the first successful positional cloning of a medaka gene (AIM1): one that encodes a transporter that mediates melanin synthesis. The protein is predicted to consist of 12 transmembrane domains and is 55% identical to a human EST of unknown function isolated from melanocytes and melanoma cells. We also isolated a highly homologous gene from the mouse, indicating a conserved function of vertebrate melanogenesis. Intriguingly, these proteins have sequence and structural similarities to plant sucrose transporters, suggesting a relevance of sucrose in melanin synthesis. Analysis of AIM1 orthologs should provide new insights into the regulation of melanogenesis in both teleosts and mammals.

Genomic analysis of gamma-ray-induced germ-cell mutations at the b locus recovered from the medaka specific-locus test.

To study how gamma-ray-induced germ-cell mutations are fixed at the early embryonic stage of the next generation, genomic alterations in the b locus mutants (colorless melanophores) detected during development in the medaka specific-locus test (SLT) were analyzed. First, nine anonymous DNA markers linked to the b locus were cloned and mapped into the region extending about 47cM surrounding the b locus. Next, losses of paternal alleles of these DNA markers were examined in each of the 51 gamma-ray-induced b locus mutants obtained after irradiation of sperm or spermatids. In these mutants, 47 were dominant lethals, three were semi-viable and one was viable. All the mutants examined had large deletions surrounding the b locus. One viable mutant had an interstitial deletion, while all the semi-viable and dominant lethal ones appeared to have terminal deletions. Deletions extending about 20-35cM were the most frequently observed in 18 of the 51 mutants examined. The largest one extended more than 40cM. These results suggest that most of the gamma-ray induced germ cell mutations recovered as total specific-locus mutants were accompanied by large genomic deletions, which eventually led the mutant embryos to dominant lethality.

A detailed linkage map of medaka, Oryzias latipes: comparative genomics and genome evolution.

We mapped 633 markers (488 AFLPs, 28 RAPDs, 34 IRSs, 75 ESTs, 4 STSs, and 4 phenotypic markers) for the Medaka Oryzias latipes, a teleost fish of the order Beloniformes. Linkage was determined using a reference typing DNA panel from 39 cell lines derived from backcross progeny. This panel provided unlimited DNA for the accumulation of mapping data. The total map length of Medaka was 1354.5 cM and 24 linkage groups were detected, corresponding to the haploid chromosome number of the organism. Thirteen to 49 markers for each linkage group were obtained. Conserved synteny between Medaka and zebrafish was observed for 2 independent linkage groups. Unlike zebrafish, however, the Medaka linkage map showed obvious restriction of recombination on the linkage group containing the male-determining region (Y) locus compared to the autosomal chromosomes.

Sex-Linked Inheritance of the lf Locus in the Medaka Fish (Oryzias latipes).

The lf (leucophore free) locus was previously reported autosomal recessive in the medaka fish (Oryzias latipes). However, extensive linkage analyses in this study using various strains revealed that the lf locus was closely sex-linked. The recombination frequency between lf and the male determining factor (y) was 2.2% (10 recombinants out of 464 progeny). Because the lf/lf homozygous fish do not have visible leucophores, they are distinguishable from wild type in early developmental stages. In the Qurt strain with heterozygous sex chromosomes (X(lf)/X(lf) in females and X(lf)/Y(+) in males), we can predict sex of each embryo on second day after fertilization. The strain should be a very useful material for studying sex determination or differentiation mechanisms in the medaka fish.

Neurotransmitters partially restore glucose sensitivity of insulin and glucagon secretion from perfused streptozotocin-induced diabetic rat pancreas.

To elucidate the mechanisms of insensitivity of hormone secretion to glucose in streptozotocin-induced diabetic rat islets, we investigated the effects of acetylcholine (ACh) and norepinephrine on insulin and glucagon secretion in response to changes in glucose concentration, using perfused pancreas preparations. Basal insulin secretion at a blood glucose level of 5.6 mmol/l was significantly higher and basal glucagon secretion significantly lower in streptozotocin-induced diabetic rats than in controls, and neither high (16.7 mmol/l) nor low (1.4 mmol/l) blood glucose concentrations influenced insulin or glucagon secretion. Addition of 10(-6) mol/l ACh to the perfusate increased glucose-stimulated insulin secretion. Also, 10(-6) mol/l ACh, 10(-7) mol/l norepinephrine, as well as a combination of both, induced marked glucagon secretion, this was suppressed by high blood glucose level. Although simultaneous addition of 10(-6) mol/l ACh and 10(-7) mol/l norepinephrine induced only a slight increase in glucagon secretion in response to glucopenia, there was a significant increase in glucagon secretion in conjunction with an ambient decrease in insulin. Histopathological examination revealed a marked decline in acetylcholinesterase and monoamine-oxidase activities in the islets of streptozotocin-induced diabetic rats. We speculate that reduction of the potentiating effects of ACh and norepinephrine lessens glucose sensitivity of islet beta and alpha cells in this rat model of diabetes.

Cholangiocarcinoma arising in bile duct adenoma with focal area of bile duct hamartoma.

A 59-year-old male with history of sigmoid colon cancer had a high serum-CEA level and was referred for the evaluation of metastatic liver disease. Ultrasonography and computerized tomography showed two tumours in the liver. Macroscopically, these were in segment 4 (S4) and 2 (S2). Histologically, the tumour in S4 showed a number of bile ductules with variable amounts of stroma, an appearance compatible with bile duct adenoma (BDA). There were markedly atypical ductules of various sizes, the epithelium of which had coarsely granular/hyperchromatic large nuclei, in some areas of the lesion. These atypical ductules showed invasive growth into the liver parenchyma. Some cystically dilated ductules with bile plugs resembling bile duct hamartoma (BDH) were also seen. The other tumour in S2, was a metastatic adenocarcinoma from sigmoid colon and showed strongly positive staining for CEA. Since the lesion in S4 of our case is solitary and most of histological features are similar to those of BDA with markedly atypical bile ductules, we consider that this may be the first case of cholangiocarcinoma associated with BDA with focal area of BDH. It is possible that the adenoma-carcinoma sequence occurs in biliary tumours.

Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

The significance of relationship of the presence of nonspecific suppressor cells in spleens with gastric cancer-related pathology.

Lymphocyte-rich mononuclear cells (MC) were prepared from spleens removed at the time of surgery for gastric cancer and the suppressor cell activity on autochthonous peripheral blood lymphocyte (PBL) responses to PHA-P and Con A was assessed. Suppressor cell activity of MC on PBL responses to at least one mitogen, either PHA-P or Con A, was observed in 52 percent (11/21) of the gastric tissues studied. MC of patients with stage IV disease appeared to exhibit the highest frequency of the presence of the activity. MC of patients with cancer with microscopically proven metastasis to group 2 lymph nodes or to more distant lymph nodes were preponderant in showing the activity. MC of patients with cancer occupying the greater curvature or the entire circumference of the stomach were also preponderant in the activity. The implications of these findings are discussed.

[Clinical experience with cefoxitin in the surgical field].

Cefoxitin (CFX) was administered to 49 hospitalized patients in the surgical field, and the following results were obtained: Clinical results of the 12 patients with surgical infections were excellent in 1 patient, good in 10, and poor in 1, with the efficacy rate of 91.7%. CFX was also administered to 37 patients for prophylaxis of postoperative infections, and the clinical efficacy rate was 91.7%. No side effects were seen besides mild eruption in 1 patient. The above results indicate that CFX is exceeding useful in surgical field.

[Effect of splenic mononuclear cells on autochthonous peripheral blood lymphocyte responses to mitogens in gastric cancer patients].

Mononuclear cells and their fractions which were prepared from spleens removed at the time of surgery for gastric cancer were treated with mitomycin C and their effect on autochthonous peripheral blood lymphocyte responses to PHA-P and Con A was assessed in coculture experiments. Splenic mitomycin C treated mononuclear cells from certain gastric cancer patients were found to have suppressor activity on peripheral blood lymphocyte responses to PHA-P and Con A and the cells with the activity were either plastic dish adherent cells or T-enriched cells alone or both of them. The suppressor activity were more frequently detected in patients with stage IV disease and the patients with the activity tended to exhibit more decreased cell mediated immunocompetence than those without it. Splenic mitomycin C treated mononuclear cells from certain gastric cancer patients were also found to have augmenting activity on peripheral blood lymphocyte responses to PHA-P and the cells with the activity were either plastic dish adherent cells or T-enriched cells or all of plastic dish adherent cells and T- and B-enriched cells.

Effect of spleen-derived cells on autochthonous peripheral blood lymphocyte responses to mitogens in gastric cancer patients.

Lymphocyte-rich mononuclear cells and their fractions which were prepared from spleens removed at the time of total or proximal gastrectomy for gastric cancer were treated with mitomycin C, and their effect on autochthonous peripheral blood lymphocyte responses to phytohemagglutinin P and concanavalin A was assessed in coculture experiments. It was found that splenic mitomycin C-treated lymphocyte-rich mononuclear cells from certain gastric cancer patients had suppressor cell activity on peripheral blood lymphocyte responses to phytohemagglutinin P and concanavalin A and that the cells with the activity were either plastic dish-adherent cells or T-enriched cells alone or both of them. It was also found that splenic lymphocyte-rich mononuclear cells from certain gastric cancer patients had augmenting activity on peripheral blood lymphocyte responses to phytohemagglutinin P and that the cells which exerted the activity were either plastic dish-adherent cells or T-enriched cells or all of plastic dish-adherent cells and T- and B-enriched cells.

Biliary cystadenocarcinoma with peritoneal carcinomatosis.

An autopsy report of a patient with a hugh biliary cystadenocarcinoma arising in the right lobe of the liver is presented. The tumor had been though to be benign, but peritoneal carcinomatosis developed 18 months after the operation. Autopsy revealed marked peritoneal dissemination of mucinous adenocarcinoma, apparently a result of peritoneal seeding of hepatic cystadenocarcinoma, which was presumed to be triggered by cyst aspiration performed during the operation. Experience indicates that surgical procedures on cystic liver disease should be carried out carefully, considering the possible existence of malignancy.

[On the leptospirosis occurred in a worker under construction of World Ocean Exposition in Okinawa Prefecture (author's transl)].

A male worker, 29 years old, was engaged in construction work of World Ocean Exposition in Okinawa Perfecture, and was diagnosed as suffering from leptospirosis in September 1973. The leptospirosis was an acute infection caused by Leptospira hebdomadis. The parasite seemed to be transmitted to him by ingestion of food and drink contaminated with the urine and excreta of the reservoir animals in the unsanitary workshop and living environment in Okinawa district. This leptospirosis was determined as an occupational disease.

Synthesis of ribonucleic acid by isolated rat liver mitochondria.

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.