Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

Roles of neonatal and prepubertal testicular androgens on androgen-induced proliferative response of seminal vesicle cells in adult mice

Male mice were castrated at 0, 10, 20, 30, 40 and 60 days of age; daily injections of testosterone propionate (TP, 4 micrograms/g b. wt) were started from day 90. On various days after starting the TP injections, the incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was determined as an index for proliferation. The seminal vesicle cells in mice castrated on days 0 and 20 were characterized by low weight (0.5-1 mg) before TP injection, long duration of androgen-induced proliferation (greater than 20 days) with a low peak, and involvement of both epithelial and fibromuscular cells (neonatal castration type). The seminal vesicle cells in mice castrated on days 60 and 40 were characterized by relatively high weight (5-10 mg) before TP injection, short duration of androgen-induced proliferation (10 days) with a high peak, and involvement of only the epithelial cells (adult castration type). In mice castrated on days 0 and 20, the neonatal castration type of androgen-induced proliferation was completely changed to the adult castration type when TP pretreatment (2 micrograms/g b. wt per 12 h) had been given from day 20 to day 40. However, the TP pretreatment given from day 90 to day 110 instead of days 20-40 had no such effect in 140-day old mice castrated on day 0. The present findings suggest that testicular androgens secreted from day 20 to day 40 play an indispensable role in the induction of irreversible proliferative response of the mouse seminal vesicle. The activity of the prepubertal androgens may not be completely compensated by androgen activity at adulthood.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

Effect of lard and corn oil intake on serum lipids in young men

An experimental diet with lard (30 g/day for 7 days) and corn oil (30 g/day for 7 days) on high carbohydrate (basal diet) was given to four healthy Japanese young men and the effect of diets containing different fat on serum lipids was examined. Serum total cholesterol was increased significantly from a basal diet of 106 +/- 23 to 141 +/- 26 mg/dl on lard diet, and then decreased significantly (p less than 0.05) to 111 +/- 22 mg/dl on corn oil diet. Serum triglycerides increased significantly (p less than 0.01) from 66 +/- 38 to 173 +/- 32 mg/dl on basal diet. Serum HDL-cholesterol was decreased significantly (p less than 0.01) from 41.9 +/- 1.6 to 31.2 +/- 3.8 mg/dl on lard diet and increased significantly (p less than 0.05) to 41.9 +/- 4.6 mg/dl on corn oil diet. Serum HDL-cholesterol fraction was decreased significantly (p less than 0.01) from 41.6 +/- 4.9 to 28.1 +/- 3.2% on basal diets, but increased significantly (p less than 0.05) to 44.3 +/- 3.1% on lard diet, and then decreased to 36.3 +/- 2.5% on corn oil diet. Serum HDL phospholipid fraction decreased significantly (p less than 0.05) from 62.5 +/- 6.7 to 50.7 +/- 1.8% on basal diet and increased significantly (p less than 0.05) to 60.4 +/- 1.0% on lard and corn oil diet. Serum phospholipids did not change by experimental diets. It is concluded that lard and corn oil have different and specific roles in lipid metabolism.     

Roles of prepubertal androgen, estrogen or androgen plus prolactin on androgen-induced proliferative response of seminal vesicles in adult mice

Male mice castrated on day 0 after birth were pretreated daily with testosterone propionate (TP, 4 micrograms/g body weight), 17 beta-estradiol (E2, 0.2 micrograms/g body weight) or vehicle for 21 days starting from day 20. In another experiment, male mice were castrated on day 25; two pituitaries from 60-day-old females were immediately grafted under the capsule of the left kidney in one group. The castrated mice with or without grafts were pretreated daily with TP (4 or 20 micrograms/g body weight) for 36 days starting from day 25, and the left kidney was removed on day 60. Daily TP injections (4 micrograms/g body weight) were started again at 30 days after the end of pretreatments to examine androgen-induced proliferation, and incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was used as an index of proliferation. In the neonatally castrated mice, both TP and E2 pretreatments given during the prepubertal period significantly increased seminal vesicle weight even long after the end of the pretreatments. However, androgen-induced proliferative response found in the neonatally castrated adult mice (poor response; long duration with a low peak) was changed to that found in mice castrated at adulthood (good response; short duration with a high peak) by the TP pretreatment only but not at all by the E2 pretreatment. In the mice castrated on day 25, a pharmacological dose of TP or TP plus hyperprolactin could not enhance or change the adult castration type of androgen-induced proliferation induced by physiological prepubertal androgens, although both treatments significantly enhanced the prepubertal growth of the seminal vesicles.     

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system

Summary The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobueet al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra-and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin.The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid