Degradation of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase in the Lysates of the Chloroplasts Isolated Mechanically from Wheat (Triticum aestivum L.) Leaves

Intact chloroplasts were isolated mechanically from the primaryleaves of 8- to 12-day old seedlings of wheat (Triticum aestivumL.) and purified by Percoll gradient centrifugation. The chloroplastswere lyzed by osmotic shock and the reaction mixtures containingthe lysates were incubated in the pH range of 5.3 to 9.4 at37°C. The degradation of ribulose-l,5-bisphosphate carboxylase/oxygenase(RuBisCO, EC 4.1.1.39 [EC] ) and its degradation products in the mixtureswere examined by using SDS-polyacrylamide gel electrophoresis.RuBisCO-hydrolase activity in the lysates was very weak, andit was difficult to assess the activity by measuring the lossof the amount of the large subunit of RuBisCO on the gels afterstaining with Coomassie Brilliant Blue. By using immunoblottingmethod, however, degradation products of RuBisCO could be detectedin the reaction mixtures. The hydrolase activity was pronouncedin the presence of 0.1 % (w/v) of SDS in the reaction mixtures.Among the products, the 35 kDa fragment was conspicuous andfound in the wide range of pHs. This degradation of RuBisCOwas inhibited in the presence of leupeptin and N-ethylmaleimide. (Received October 3, 1988; Accepted November 25, 1988)     

Anti-diabetic effects of globin digest and its active ingredient Leu-Ser-Glu-Leu in ICR mice,streptozotocin-induced diabetic mice and KK-Ay mice

AimsLeu-Ser-Glu-Leu (LSEL) is the main active ingredient of globin digest (GD) that has an anti-diabetic effect. Here, we investigated the anti-diabetic effect of LSEL for the first time.Main methodsThe anti-diabetic effects of GD and LSEL in ICR mice, streptozotocin (STZ)-induced diabetic mice and KK-Ay mice were examined.Key findingsGD and LSEL suppressed the elevation of blood glucose in an oral glucose tolerance test (OGTT) in ICR mice, STZ-induced diabetic mice and KK-Ay mice as well as in an oral sucrose tolerance test in ICR mice and in an insulin tolerance test (ITT) in KK-Ay mice. GD and LSEL decreased the blood glucose levels in the basal state in STZ-induced diabetic mice and KK-Ay mice. Furthermore, GD and LSEL elevated the serum insulin levels in an OGTT in ICR mice and KK-Ay mice and promoted the use of insulin in an ITT in KK-Ay mice. GD and LSEL increased the translocation or expression of the glucose transporter 4 in the muscle of ICR mice, STZ-induced diabetic mice and KK-Ay mice and increased the expression of the uncoupling protein 2 (UCP2) in the muscle of ICR mice.SignificanceThese results indicate that GD and LSEL control blood glucose through the promotion of glucose uptake in the muscle of the mice. The acceleration of glucose uptake by GD and LSEL may be controlled by the promotion of insulin secretion and the up-regulation of UCP2 expression. GD and LSEL seem to be useful for lowering the incidence of hyperglycemia.     

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha 2 (GPA2) and beta 5 (GPB5)

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.     

Hypomethylation of paternal DNA in the late mouse zygote is not essential for development

Global demethylation of DNA which marks the onset of development occurs asynchronously in the mouse; paternal DNA is demethylated at the the zygote stage, whereas maternal DNA is demethylated later in development. The biological function of such asymmetry and its underlying mechanisms are currently unknown. To test the hypothesis that the early demethylation of male DNA may be associated with protamine-histone exchange, we ,used round spermatids, whose DNA is still associated with histones, for artificial fertilization (round spermatid injection or ROSI), and compared the level of methylation of metaphase chromosomes in the resulting zygotes with the level of methylation in zygotes obtained after fertilization using mature sperm heads (intracytoplasmic sperm injection or ICSI). In contrast to ICSI-derived zygotes, ROSI-derived zygotes possessed only slightly demethylated paternal DNA. Both types of zygotes developed to term with similar rates which shows that hypomethylation of paternal DNA at the zygotic metaphase is not essential for full development in mice. Incorporation of exogenously expressed histone H2BYFP into paternal pronuclei was significantly higher in ICSI-derived zygotes than in ROSI-derived zygotes. Surprisingly, in the latter the incorporation of histone H2BYFP into the paternal pronucleus was still significantly higher than into the maternal pronucleus, suggesting that some exchange of chromatin-associated proteins occurs not only after ICSI but also after ROSI. This may explain why after ROSI, some transient demethylation of paternal DNA occurs early after fertilization, thus providing support for the hypothesis regarding the link between paternal DNA demethylation and protamine/histone exchange.     

The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.     

Degradation of 4′-dimethylaminoazobenzene-2-carboxylic acid by Pseudomonas stutzeri

4-Dimethylaminoazobenzene-2-carboxylic acid (DMBC) was utilized as a necessary carbon and nitrogen source by Pseudomonas stutzeri IAM 12097. o-Aminobenzoic acid (o-ABA), N,N-dimethyl-p-phenylenediamine (DMPA) and cathecol were identified as intermediates of DMBC degradation. DMBC was degraded at a concentration below 70 mol dm^–3. The ability to utilize DMBC in P. stutzeri was lost spontaneously to some extent. When P. stutzeri was cured of plasmid DNA (approximately 8 MDal) by treatment with mitomycin C, acridine orange, and chloramphenicol, DMBC was not utilized by the resultant strain. These facts suggest that the degradative ability on DMBC in P. stutzeri is controlled by plasmid DNA. Correspondence to: C. Yatome     

Conditions of establishment for the Salix community at lower-than-normal water levels along a dam reservoir shoreline

In limited-water-level reservoirs, areas along the reservoir shoreline are often exposed within the drawdown zone when water levels are reduced from normal to limited levels during the flood season. To prevent erosion and conserve the landscape, test plantings and landscaping along the shoreline have been implemented at some dam reservoirs. The establishment of vegetation within the drawdown zone contributes to both landscape and wildlife habitat conservation. Distribution, habitat, and seed-dispersal period of willows such as Salix subfragilis were investigated at Miharu Dam reservoir in northeastern Japan. Eight willow species were found around the dam, but S. subfragilis dominated within the drawdown zone. Field survey results beginning in 1995 indicate that the size of the S. subfragilis community increased after initial impoundment. Although many other Salix species disperse seeds prior to the drawdown period, S. subfragilis disperses during and after drawdown in the spring; thus, when water levels are lower than normal, its seeds are supplied to the newly exposed zones that provide suitable habitat for Salix seedling establishment. Our results suggest that S. subfragilis dominated because of water-resistant properties and timing of the seed dispersal period.     

Overexpression of stearoyl-coenzyme A desaturase 1 in macrophages promotes reverse cholesterol transport

Stearoyl-coenzyme A desaturase 1 (SCD1) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. However, the impact of SCD1 on atherosclerosis remains unclear. The aim of this study was to determine whether SCD1 affects macrophage reverse cholesterol transport (RCT) in mice. Compared to the control, adenoviral-mediated SCD1 overexpression in RAW264.7 macrophages increased cholesterol efflux to HDL, but not to apoA-I, without clear changes in ABCA1, ABCG1 and SR-BI expressions. While knockdown of ABCG1 and SR-BI did not affect the SCD1-induced cholesterol efflux to HDL, SCD1-overexpressing macrophages promoted the formation of both normal- and large-sized HDL in media, accompanying increased apolipoprotein A-I levels in HDL fractions. Transformation to larger particles of HDL was independently confirmed by nuclear magnetic resonance-based lipoprotein analysis. Interestingly, media transfer assays revealed that HDL generated by SCD1 had enhanced cholesterol efflux potential, indicating that SCD1 transformed HDL to a more anti-atherogenic phenotype. To study macrophage RCT in vivo, ³H-cholesterol-labeled RAW264.7 cells overexpressing SCD1 or the control were intraperitoneally injected into mice. Supporting the in vitro data, injection of SCD1-macrophages resulted in significant increases in ³H-tracer in plasma, liver, and feces compared to the control. Moreover, there was a shift towards larger particles in the ³H-tracer distribution of HDL fractions obtained from the mice.