Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.     

Causes of chromosome anomalies suggested by cytogenetic epidemiology of induced abortions

Summary Women who visited hospitals requesting induced abortions were asked about their obstetric and medical histories by attending physicians prior to the operation. The information was solely based on their reports at the interview. The period of coverage was nine weeks, comprising six weeks before and three weeks after the onset of the last menstruation. Both the occurrence of acute respiratory infectious diseases, which included the common cold, upper respiratory tract infection (U.R.T.I.), influenza, and pneumonia, and the use of analgesics and antipyretics were most frequently reported. Regardless of whether they had an acute respiratory illness, the women who took analgesics and antipyretics (including antihistamine, cough remedy, and nasal decongestant) had 13.8% (8/58) gross chromosome anomalies. The women without the treatment had 6.3% (71/1136) anomalies. The difference was statistically significant (P<0.05). Triploid formation was particularly related to drug consumption, thus there were 5.2% (3/58) in the treated group and 0.5% (6/1136) in the untreated group (P=0.0074). In contrast, trisomy was not considered to be related to drug consumption on account of 8.6% (5.58) and 4.1% (47/1136) in the corresponding groups (0.10<P<0.20). When the occurrence of trisomy was examined in connection with two variables, i.e., the presence of drug consumption and the age of mothers, association was not evident (₂ assoc.=0.883,d.f.=1 by Cochran.s method). Besides these results, neither maternal X-irradiation nor irregularity of the menstrual period was found to be related to the frequency of chromosome anomalies in the progeny.     

Studies of isoniazid metabolism in isolated rat hepatocytes by mass fragmentography

Isoniazid metabolism in isolated rat hepatocytes was studied by mass fragmentography using single ion monitoring. Isoniazid and its metabolites were determined as the trimethylsilylated derivatives of acetylisoniazid and diacetylhydrazine and of the benzaldehyde hydrazones of isoniazid and acetylhydrazine. Deuterated analogues served as internal standards. Hydrazine was quantitated as benzalazine using ¹⁵N-labeled hydrazine as an internal standard. The method is well suited for the microanalysis of isoniazid metabolites in specificity and reliability to demonstrate the overall pathway of isoniazid metabolism, from which it was clarified that the greater part of hydrazine, a hazardous metabolite of isoniazid, was formed through the direct hydrolysis of isoniazid itself as expected.     

Isolation of Crystalline Water-soluble Chlorophyll Proteins with Different Chlorophyll a and b Contents from Stems and Leaves of Lepidium virginicum

Two types of water-soluble chlorophyll proteins were isolatedfrom Lepidium virginicum L. grown in Japan. The protein isolatedfrom the leaves (CP663L) had a low chlorophyll a/b ratio (1.5–1.7),and that from the stems (CP663S) had a high ratio (3.4–3.5).CP663S showed the same crystal forms and almost the same molecularweight and subunit composition as CP663I. (Received October 26, 1981; Accepted February 4, 1982)     

Geographical distribution of Phagocata vivida in the Far East

Phagocata vivida (Ijima et Kaburaki, 1916) is common in cold-water habitats in mountainous and hilly regions in Japan; in Northern Japan it occurs in lowland areas. Comparative studies of the material from South Korea and Primorskiy in Northeast Siberia, Russia, show that Ph. vivida is distributed widely in these geographic areas. Phagocata miyadii Okugawa, 1939, reported from North Korea and Northeastern China is a synonym of Ph vivida. Geographical distribution of this species in the Japanese Islands now becomes very clear. Judging by its geographical and vertical distributions, the species probably is a preglacial faunal element that entered Japan by the northern route to Old Honshû Island along the coast of the Old Sea of Japan.     

A review of chromosomal variation in Dugesia japonica and D. ryukyuensis in the Far East

The chromosome numbers of Dugesia japonica Ichikawa et Kawakatsu, 1964, are n = 8, 2x = 16 and 3x = 24; those of Dugesia ryukyuensis Kawakatsu, 1976, are n = 7, 2x = 14 and 3x = 21. The karyotypes of both species include diploid, triploid and mixoploid; aneuploidic and mixoaneuploidic karyotypes may occur. In 785 specimens studied of D. japonica, the occurrence rates of specimens having each karyotype are substantially the same (29–37%). Diploid sexual specimens represented nearly 10% of the total and virtually no triploid or mixoploid sexual specimens were found. The diploid karyotype can be inherited by both sexual and asexual reproduction; the triploid and mixoploid karyotypes will be inherited only by asexual reproduction. In 51 specimens studied of D. ryukyuensis, the different karyotypes are diploid (ca 39%), triploid (ca 57%) and mixoploid (ca 4%). Diploid sexual specimens represented nearly 25% of the total; sexual specimens with tripooidic karyotypes made up nearly 27%. The diploid, triploid and mixoploid karyotypes were also found in juveniles hatched from cocoons. The diploid karytyype is inherited by both sexual and asexual reproductions; the other karyotypes may be inherited by parthenogenesis or self-fertilization (including pseudogamy) and asexual reproduction.     

Developmental Potentials of Early Telencephalic Neuroepithelial Cells: A Study with Microexplant Culture

To gain a better understanding of how neuron generation begins in the telencephalon, we analyzed the developmental potentials of neuroepithelial cells (NE cells) using a microexplant culture system. By the patterns of neuron generation and cell spreading on fibronectin (FN) in vitro , we recognized at least three different types of NE cells in the early (E9 to E10.5) telencephalon of mice. Although there was no transition from one growth pattern to another in vitro , these patterns were found to represent distinct stage-dependent profiles of the NE cells. (i) The youngest type of NE cells spread well and formed flat cell sheets on FN, but generated no neurons at all. (ii) An intermediate type showed extensive spreading on FN and generated a small number of neurons only early in culture. (iii) In contrast, NE cells at the most advanced stage examined formed compact cell sheets independent of FN and showed continuous neuron generation. These results indicate that telencephalic NE cells at the beginning of neurogenesis do not have a potential to make all cerebral neurons simultaneously. Our findings suggest that, during the period of E9 to E10.5, NE cells first acquire a potential to make the neurons produced before E12.5 and the NE cells then become capable of generating the neurons formed later under the influence of environmental factors.     

Protein tailoring of human granulocyte colony-stimulating factor

Summary Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was modified by site-directed mutagenesis and chemical modification in order to improve its pharmacological activity and its thermostability. The mutant rhG CSF which 17th cysteine was substituted with alanine was chemically modified by activated polyethylene glycol. The chemically modified mutant rhG-CSF greatly increased both its biological activityin vivo and its thermostability. This is a successful example of protein tailoring in which site-directed mutagenesis and chemical modification were used at the same time.     

Amino acid sequence of trichoanguina,a ribosomal-inactivating protein fromTrichosanthes anguina seeds

In this study, we sequenced a new type I ribosome-inactivating protein, trichoanguina, from the seeds ofTrichosanthes anguina (snake gourd). Trichoanguina is a basic glycoprotein having an apparent molecular mass of 35.0 kD and possessing strong ribosome-inactivating activity. Trichoanguina was cleaved with cyanogen bromide and partially digested with thermolysin, chymotrypsin, trypsin andStaphylococcus aureus V8 protease. The subsequent peptide fragments were separated by SDS-polyacrylamide gel electrophoresis, followed by electroblotting to polyvinylidene difluoride membranes and then sequencing. The sequencing of trichoanguina was completed, consisting of 245 amino acid residues. The sequencing of trichoanguina revealed a considerable homology to trichosanthin anda-trichosanthin, which are known as abortifacient, ribosome-inactivating and antihuman immunodeficiency virus proteins, with 46.7% and 55.6% amino acid identities, respectively. The sequence conserves two active sites: Glu-158 and Arg-161.     

β-Galactosidase-deficient mouse as an animal model for GM1-gangliosidosis

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis. This revised version was published online in November 2006 with corrections to the Cover Date.