Duplication of an amphioxus myogenic bHLH gene is independent of vertebrate myogenic bHLH gene duplication.

Gene duplication is thought to be a major genetic change that may have permitted the evolution of vertebrates from invertebrates. The myogenic genes encode basic helix-loop-helix (bHLH) transcriptional factors essential for the formation of skeletal muscle. The invertebrate genome contains only a single myogenic bHLH gene, whereas the vertebrate genome contains four (MyoD, Myf-5, myogenin and MRF4). Since the tunicate genome contains a single myogenic bHLH gene, its duplication might have occurred some time during chordate evolution. To determine whether the duplication of the myogenic bHLH gene occurred prior to, or after the divergence of vertebrates from the cephalochordate lineage, we amplified target fragments from the amphioxus, Branchiostoma floridae, by means of PCR. Sequence analysis and genomic Southern analysis revealed that the amphioxus genome contains two myogenic bHLH genes (BMD1 and BMD2). A comparison of the amino acid sequences in the bHLH domain between BMD1, BMD2 and four vertebrate myogenic bHLH gene products, however, showed that neither BMD1 nor BMD2 resembled any of the four genes. These results suggested that the duplication of amphioxus myogenic bHLH gene occurred independently of that leading to the four myogenic bHLH genes in vertebrates.

Radioimmunoassay for pyrazolone derivatives.

The determination of pyrazolone derivatives by radioimmunoassay has been reported. Anti-antipyrine antisera were obtained by repeated immunization of rabbits with 4-azoantipyrine-conjugated bovine serum albumin. The level at least as low as 1.0 ng of antipyrine could be detected by this procedure. Various substituents on the carbon 4 position of the pyrazolone ring as well as the lack of methyl group on the nitrogen 2 position decreased the affinity for the antibody, and the antibody showed no cross-reaction with any pyrazolidine derivatives.

Immunohistochemical localization and dynamics of paraquat in small intestine, liver and kidney.

Immunohistochemical techniques were used to observe the localization of paraquat in the small intestine, liver and kidney, organs that absorb and eliminate chemicals. Paraquat-poisoned rats were killed 3 h, 12 h, 24 h, 3 days, 7 days and 10 days after intravenous administration of paraquat. Three hours after injection, paraquat was localized in hepatocytes and in the kidney in the epithelial cells of the distal tubule. The amount of paraquat in the liver and kidney increased by 24 h after the administration and thereafter decreased with time, suggesting that paraquat is secreted into bile and urine. In the intestine, 3 h after injection, paraquat was localized in the epithelial cells. The same finding was also made in rats with a cannulated bile duct. Therefore, it is likely that paraquat is secreted into the gut lumen from epithelial cells and that paraquat secreted from liver into the duodenum is reabsorbed into the epithelial cells of the intestine.

Isolation of intact plankton from drowning lung tissue by centrifugation ina colloidal silica gradient.

Several species of intact phyto- and zooplankton from the homogenate of rat "drowning lung" were separated by centrifugation in a colloidal silica gradient. The plankton, except diatoms, were isolated from a small amount of human "drowning lung" by this procedure. This new method was found to be more useful in detecting plankton in tissues than chemical digestion with strong acid.

Estimation of stature from somatometry of skull.

In order to investigate the possibility of estimating stature from somatometry of the skull, we carried out a study on 124 Japanese cadavers (comprising 77 males and 47 females) that had been autopsied at our laboratory between July 1986 and June 1991. Somatometry of the skull was performed on diameter (distance between glabella and external protuberance) and circumference (length around the skull through two points: the glabella and the external protuberance). The regression equations calculated were as follows: stature in males = (diameter + circumference) x 1.35 + 70.6 (standard error of estimate (S.E.) = 6.96 cm); stature in females = circumference x 1.28 + 87.8 (S.E. = 6.59 cm); stature in both sexes = (diameter + circumference) x 1.95 + 25.2 (S.E. = 7.95 cm). These S.E.s appear to be larger than those obtained for other parts of the body. However, in cases where identification is required by means of only the skull, this method could prove useful.

Identification of 10-hydroxy-12-octadecenoic acid in adipocere.

Studies are reported on separation and identification of 10-hydroxy-12-octadecenoic acid in human adipocere. The chemical structure was determined by thin-layer chromatography (TLC), gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS).

The mechanism of experimental adipocere formation: hydration and dehydrogenation in microbial synthesis of hydroxy and oxo fatty acids.

The enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxystearic acid but dehydrogenation of this product. The mechanism of the hydration and dehydrogenation was proved by gas chromatography-mass spectrometry of 10-hydroxy and 10-oxostearic acids produced in the presence of D2O or H2(18)O. The activity of these enzymes was increased by preincubating Flavobacterium meningosepticum with oleic acid.

The mechanism of experimental adipocere formation: substrate specificity on microbial production of hydroxy and oxo fatty acids.

Studies are reported on microbial conversion of various unsaturated fatty acids to 10-hydroxy and/or 10-oxo fatty acids by Micrococcus luteus. Four fatty acids possessing cis-9-unsaturation produced 10-hydroxy and 10-oxo fatty acid products, but three enoic acids possessing trans-9-unsaturation or double bond(s) in other than the 9-carbon position were inactive as substrates. 10-Hydroxy palmitic and stearic acids were converted to the corresponding 10-oxo fatty acids, but the 10-oxo compounds were inactive as substrates. This indicates that the metabolic sequence of cis-9-enoic fatty acid by the microbial enzyme(s) is first converted to 10-hydroxy fatty acid and then to its 10-oxo compound.

Lewis typing of human saliva stains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies.

The Lewis blood grouping of human saliva stains could be detected by an enzyme-linked immunosorbent assay (ELISA) using anti-Le(a) and anti-Le(b) monoclonal antibodies with an avidin-biotin complex (ABC). The saliva stains (1.0 by 1.0 cm in size) were used as samples and not only could the Lewis substances of 57 individual stains be correctly typed by this method, but also it was clarified that there are several different secretion patterns of amounts of Le(a) and Le(b) substances in 3 individual Lewis types.

The evaluation of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) by oral administration in cynomolgus monkeys in a 13 week subacute toxicity study.

To assess the subacute toxicity of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) a 13 week subacute toxicity study by gavage was done in Cynomolgus monkeys at dosage levels of 0, 15, 45 and 135 mg/kg/day. Deaths were seen in the 135 mg/kg/day group; with associated debility. The animals that died had high plasma levels of FUT-187. Little weight gain was seen in the 135 mg/kg/day group. There were no clear treatment related effects on ophthalmoscopy or electrocardiography or on hematological, biochemical or urinalysis. There were no effects noted at necropsy or on histopathology and the causes of death for each of the 3 animals that died were suppurative glomerulonephritis, bone marrow depression and possible gavage injury respectively. The no-effect level for toxicology was considered to be 45 mg/kg/day.

[Acute toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187) in mice, rats and dogs.

Single oral, subcutaneous or intravenous administration to mice and rats and oral administration to dogs were performed to investigate the acute toxicity of FUT-187. 1) LD50 values in mice were 4,395 mg/kg for males and 3,626 mg/kg for females orally, 6,284 mg/kg for males and 5,492 mg/kg for females subcutaneously, and 39.4 mg/kg for males and 41.4 mg/kg for females intravenously. In rats, these values were 4,653 mg/kg for males and 3,761 mg/kg for females orally, 6,799 mg/kg for males and 3,343 mg/kg for the females subcutaneously and 21.8 mg/kg for males and 15.8 mg/kg for females intravenously. 2) Death occurred 2 hours after administration in a male dog of the 3,000 mg/kg group just after convulsion and nasal discharge were observed. 3) General symptoms in mice and rats included a creeping gait, convulsion, singultus, cyanosis, decreased locomotor activity, piloerection and salivation which were commonly observed by all routes. All dogs showed vomiting and decreased locomotor activity; the prone or lateral position, crouching, ataxic gait and salivation were also observed in many cases. 4) On autopsy, changes attributable to local irritation by FUT-187 were seen in all species except mice and rats dosed intravenously. For the gastro intestinal-tract (GIT), inflammation of the stomach, adhesions between the stomach and the liver and sclerosis, petechiae or ulcer were observed in mice and rats dosed orally. In the subcutaneous route, retention of the test compound and necrosis at the injection site were observed. Reddening and loss of mucosal smoothness were observed in the GIT of a dog which died; desquamation, congestion, hemorrhage and retention of tested compound in the digestive mucosa were observed on histopathology.

[A 52-week oral chronic toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) in rats with a recovery period of 5 weeks.

The chronic toxicity of FUT-187, a synthetic protease inhibitor, was investigated in Sprague-Dawley rats. FUT-187 was given orally to the rats at doses of 0.4, 2, 10, 50 and 250 mg/kg/day for 52 weeks. The drug was then withdrawn for 5 weeks. The results are summarized as follows: There were no deaths or toxic signs caused by the drug throughout the experimental period. There were no drug-related changes in food consumption, ophthalmological examination, hematology or blood chemistry. Slight suppression of growth was observed in males in the 250 mg/kg group. This change was reversed on withdrawal of the drug. Drug crystals were observed in the urinary sediments of both sexes in the 250 mg/kg group, but this change disappeared on withdrawal of the drug. Gross pathological examination revealed the following changes: enlargement and nodule formation in the pancreas in both sexes given more than 10 mg/kg of the drug; dark red spots in the glandular stomach in males in the 250 mg/kg group; thickening of the small intestinal walls in both sexes given more than 50 mg/kg. Of these organs, no changes were observed in the stomach and small intestine at the end of the recovery period. Increased pancreas weight was observed in both sexes given more than 50 mg/kg of the drug. Examination at the end of the recovery period suggested reversibility, showing a lesser degree of change. Histopathological examination revealed the following changes in the pancreatic acinar cells: acidophilic foci and nodules in both sexes given more than 10 mg/kg of the drug; adenoma in one male in the 250 mg/kg group; increased zymogen granules in both sexes given more than 50 mg/kg of drug; fine vacuolization in females in the 250 mg/kg group. At the end of the recovery period, increased zymogen granules and fine vacuolization of the acinar cells were not found. Furthermore, erosion or healed erosion in the glandular stomach, duodenum and jejunum was observed in a few males or females in the 250 mg/kg group, but those changes disappeared after the recovery period. In the liver, altered cell foci was observed more frequently in males in the 250 mg/kg group than the other groups, but this change also disappeared after the recovery period. In addition, brown pigmentation in the proximal renal tubules of the kidney was observed in both sexes in the 250 mg/kg group, but lesions observed in the examination after the recovery period were less noticeable than in the examination at the end of the administration period.

[A 52-week chronic oral toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) in dogs].

A chronic oral toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187), a new protease-inhibiting agent, was carried out using male and female beagle dogs. FUT-187 was orally administered to the dogs at dose levels of 7.5, 15, 30 and 60 mg/kg/day for 52 weeks, followed by 5 weeks' recovery period. Results are summarized as follows: 1. In general conditions, vomiting, salivation and the passage of mucousy stools were observed in dogs given 15 mg/kg/day or more, and diarrhea was observed at 30 mg/kg/day or more. One male given 15 mg/kg/day showed transient pallidity of the oral mucosa, and another male in the same group showed apnea and abdominal breathing. In addition, one male given 30 mg/kg/day was euthanatized due to extreme weakness, as weight loss and pallid oral mucosa, and another male in the same group died after showing acute toxic symptoms such as hyperpnea, tonic convulsion and ataxic gait. 2. Weight gain was slightly suppressed in females given 60 mg/kg/day. No significant changes in food consumption were observed. 3. Hematological examination revealed no statistically significant changes. Decreases in RBC counts, Ht values and Hb concentrations, and increased reticulocyte counts were observed in one male of 15 mg/kg/day group, which also showed pallid oral mucosa, and in one male of the 30 mg/kg/day group, which was euthanatized in a moribund state. 4. Blood biochemistry revealed increased GPT activity in males given 15 and 30 mg/kg/day and females given 60 mg/kg/day, which was accompanied by sporadic increases in GOT, A1P and/or gamma-GTP activities. Males given 30 mg/kg/day or more showed decreased total protein. 5. Hepatic function testing (ICG test) showed no statistically significant changes. One female given 60 mg/kg/day showed increased accumulating concentration of ICG. 6. There were no toxicological changes in urinalysis, fecal occult blood, renal function (PSP clearance), ophthalmological and electro-cardiographic examinations. 7. In pathological examination, inflammatory cell infiltration and microgranuloma formation in liver were noted periportally or perivenularly in both sexes given 15 mg/kg/day or more (except for 30 mg/kg/day males). In the some cases, atrophy, degeneration and necrosis of hepatocytes and/or fibrosis around inflammatory cells and microgranuloma were observed. In the spleen, one male given 15 mg/kg/day and one female given 60 mg/kg/day showed increased plasma cells in the red pulp. In the case sacrificed in a moribund condition, findings in the liver and spleen similar to those in surviving cases were detected, but were more severe, and the liver showed diffuse fibrosis.(ABSTRACT TRUNCATED AT 400 WORDS)

[A 13-week subacute oral toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187) in rats].

The toxicity of FUT-187, a synthetic protease inhibitor, was investigated in Sprague-Dawley rats. FUT-187 was given orally to the rats at doses of 2, 10, 50, 250 and 1250 mg/kg/day for 13 weeks, then the drug was withdrawn for 5 weeks for recovery. The results are summarized as follows: In the 1250 mg/kg/day group, 9 out of 20 males died with decreased body weight and exhaustion. Histopathological examination revealed renal papillary necrosis, ulcer in the urinary bladder, hemostatic lesions in the lungs and liver, ulcer or erosion in the stomach, duodenum and jejunum. The surviving animals in this group showed swelling of the limbs due to synovitis, transient salivation immediately after administration, suppression of growth with decreased food consumption. Urinalysis revealed a low pH, increased ketones and bilirubin excretion, dark yellowish change in color, the appearance of "leaflet-shaped" crystals and increased red blood cells and epithelial cells in the urinary sediment, increased water intake, decreased specific gravity and decreased sodium, potassium and chloride in the urine. Hematologically, there was an increase in the white blood cell count. A biochemical analysis of the blood revealed decreased amylase activity, glucose and total protein levels and increased GOT activity and inorganic phosphorus levels. Pathological changes were observed in the pancreas, kidney, digestive tract, urinary bladder and liver. The pancreas showed macroscopical enlargement and increased organ weight. Histopathologically, there were several alterations in the acinar cells, such as vacuolization due to increased fat droplets, nuclear irregularity, prominent nucleoli, irregular arrangement and vesiculation of rough endoplasmic reticulum (rER), dilatation of developed Golgi apparatus and increased free ribosomes. In the kidney, increased weight and pigmentation in the proximal tubular epithelium were noted. Electron microscopically, these pigments were recognized as secondary lysosomes containing filamentous material and electron dense granules within a lucent matrix. In the digestive tract, ulcer or erosion in the stomach and duodenum, and villous proliferation in the small intestine were observed. Furthermore, hyperplasia and vacuolization were noted in the mucosal epithelium of the urinary bladder. In addition, loss of perilobular fat droplets in the liver and increased adrenal weight without histological change were observed. After a 5-week recovery period, these changes disappeared almost completely. In the 250 mg/kg/day group, slight suppression of growth the appearance of "leaflet-shaped" crystals in the urinary , sediment, increased water intake and decreased sodium in the urine were observed. The pancreas showed enlargement, increased weight, acinar cell hypertrophy with increased zymogen granules, fine vacuolization, slight derangement and vesicular of rER, and dilatation of Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)

[Antigenicity study of 6-amidino-2-naphthyl 4(-)[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) in guinea pigs and mice.

Antigenicity study of 6-amidino-2-naphthyl 4(-)[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187), a new protease inhibitor, was investigated in guinea pigs and mice and the following results were obtained. 1. In guinea pigs immunized with FUT-187 plus adjuvant by intramuscular/subcutaneous routes, ASA, ACA and PCA reactions challenged intravenously or intradermally were positive. 2. In guinea pigs immunized with FUT-187 plus adjuvant by intramuscular/subcutaneous routes, ASA and PCA reactions challenged orally were negative. 3. In guinea pigs immunized with FUT-187 by the oral route, ASA, ACA and PCA reactions were negative. 4. In guinea pigs immunized with IABA and AN plus adjuvant by intramuscular/subcutaneous routes, ASA and PCA reactions were negative. 5. 48-hr PCA reactions were elicited with sera obtained from BALB/c and C3H/He mice immunized with FUT-187 plus adjuvant by the intraperitoneal route, responses were negative. 6. From the results of hapten inhibition tests using anti-FUT-187 guinea pig serum, it is suggested that the antigenicity of FUT-187 is attributable to the its benzoic acid.

[Reproductive and developmental toxicity studies of 6-amidino-2-naphthyl 4(-)[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187). (IV)--Oral administration to New Zealand white rabbits during the period of fetal organogenesis.

Oral administration of 6-amidino-2-naphthyl 4(-)[(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethanesulfonate (FUT-187) at doses of 10, 30 and 100 mg/kg was given to New Zealand White rabbits on days 6 to 18 of gestation. The following results were obtained. Decreased food consumption and suppression of body weight gain in dams were observed and these changes contributed to the increase in aborted or prematured births and increased fetal mortality at the 100 mg/kg group. There were changes attributable to FUT-187 on external, skeletal and visceral examinations of fetuses. Based on the above, the no-effect dose level in dams and fetuses in the present study is 30 mg/kg/day.

[A 13-week subacute oral toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187) in dogs.

A subacute oral toxicity study of 6-amidino-2-naphthyl 4-[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187), a new protease-inhibiting agent, was carried out in beagle dogs of both sexes. FUT-187 was administered to dogs at daily oral doses of 15, 50 and 150 mg/kg. Dogs in 150 mg/kg group were given twice a day in a.m. and p.m.. The results were as follows: 1. Changes of physical sign attributed to FUT-187, consisted of vomiting, diarrhea, salivation, decrease of locomotor activity, sedation and hyperemia of eye mucosa. These changes expect vomiting vanished within about 2 hours after treatment. One male given 150 mg/kg died on day 19 and two females given 150 mg/kg were sacrificed on day 55 and 67 due to deterioration of systemic conditions. 2. Body weight gain was suppressed in males given 150 mg/kg and females given 50 mg/kg or more. 3. In hematological examinations, some changes suggesting anemia or inflammation were observed in a few animals received 50 mg/kg or more 4. In serum biochemical examinations, dogs given 50 mg/kg or more had decrease of albumin, total protein, A/G ratio and total cholesterol, increase of GPT activity. In liver function test, decrease of function was observed in a few animals in 150 mg/kg group. These changes diminished by the end of recovery period. 5. In autopsy findings, ulcer formation and desquamation of mucosa in the digestive tract were observed in dead or sacrificed animals and survived animals given more than 50 mg/kg. In sacrificed animals, liver was yellow in color and intussusception was seen. 6. Plasma levels of intact FUT-187 and metabolites on the day 37 or 83 were higher than that on the first day of administration. 7. In histopathological examinations, ulcer formation, desquamation, degeneration and/or atrophy of mucosa in the digestive tract were observed in the animals from 50 mg/kg and 150 mg/kg groups. In addition, fatty deposition in hepatocytes was observed in one dead animal and two sacrificed animals.(ABSTRACT TRUNCATED AT 400 WORDS)

[Reproductive and developmental toxicity study of 6-amidino-2-naphthyl 4(-)[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187). (II)--Oral administration to rats during the period of fetal organogenesis (prenatal examination)

6-Amidino-2-naphthyl 4(-)[(4,5-dihydro-1H-imidazol-2-yl) amino] benzoate dimethanesulfonate (FUT-187) was given orally to pregnant Crj : CD (Sprague-Dawley) rats from days 7 through 17 of gestation at dose levels of 50, 200 and 800 mg/kg/day. In the 800 mg/kg/day group, salivation just after dosing, suppression in body weight gain and decreased food consumption were observed. No external, visceral and skeletal anomalies attributable to FUT-187 were observed in fetuses. From the present result, it is considered that the no-effect dose level of FUT-187 for dams and fetuses are 200 mg/kg/day and 800 mg/kg/day respectively.

Role of active oxygen species in the toxic effects of glucosone on mammalian cells.

Glucosone (D-arabino-hexos-2-ulose), a typical enediol product formed both in the Maillard reaction and gamma-radiolysis of sugars, decreased survival of Chinese hamster lung V79 cells, which were incubated under MEM for 4 h. Inhibition of the decrease in cell survival by catalase and SOD suggests the role of active oxygen species, namely H2O2 and O2-, in the biological effects of glucosone. H2O2 was formed in the medium during oxidative degradation of glucosone. Inhibition of the formation of H2O2 by SOD indicates that the formation of H2O2 and the consequent decrease of the cell survival was enhanced by O2-. These results suggest that the mechanisms of the effects of glucosone on the mammalian cells in the absence of Cu2+ are different from those in the presence of Cu2+.

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b).

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.