Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO^M) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO^P) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO^P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO^P (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO^M (21%). The cytoprotective effect of the asialo-rhuEPO^P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.     

Eighteen Coral Genomes Reveal the Evolutionary Origin of Acropora Strategies to Accommodate Environmental Changes

The genus Acropora comprises the most diverse and abundant scleractinian corals (Anthozoa, Cnidaria) in coral reefs, the most diverse marine ecosystems on Earth. However, the genetic basis for the success and wide distribution of Acropora are unknown. Here, we sequenced complete genomes of 15 Acropora species and 3 other acroporid taxa belonging to the genera Montipora and Astreopora to examine genomic novelties that explain their evolutionary success. We successfully obtained reasonable draft genomes of all 18 species. Molecular dating indicates that the Acropora ancestor survived warm periods without sea ice from the mid or late Cretaceous to the Early Eocene and that diversification of Acropora may have been enhanced by subsequent cooling periods. In general, the scleractinian gene repertoire is highly conserved; however, coral- or cnidarian-specific possible stress response genes are tandemly duplicated in Acropora. Enzymes that cleave dimethlysulfonioproprionate into dimethyl sulfide, which promotes cloud formation and combats greenhouse gasses, are the most duplicated genes in the Acropora ancestor. These may have been acquired by horizontal gene transfer from algal symbionts belonging to the family Symbiodiniaceae, or from coccolithophores, suggesting that although functions of this enzyme in Acropora are unclear, Acropora may have survived warmer marine environments in the past by enhancing cloud formation. In addition, possible antimicrobial peptides and symbiosis-related genes are under positive selection in Acropora, perhaps enabling adaptation to diverse environments. Our results suggest unique Acropora adaptations to ancient, warm marine environments and provide insights into its capacity to adjust to rising seawater temperatures.     

Preparation of 8-Chloropurine Nucleosides Through the Reaction Between their C-8 Lithiated Species and p-Toluenesulfonyl Chloride

Abstract

Chlorination of purine nucleosides protected with tert-butyldimethylsilyl (TBDMS) group was examined by the reaction of the C-8 lithiated species, generated by LDA, with p-toluenesulfonyl chloride as an electrophile. This provides a new method for the preparation of 8-chloropurine nucleosides.     

Anesthetic Effects of a Mixture of Medetomidine,Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.     

P450 in wild animals as a biomarker of environmental impact

The impact of environmental pollution on selected animals was tested by monitoring the hepatic content of cytochromes P450 and their enzyme activities or by calculating TEQ values from the concentration of pollutants in the body. Fish-eating Stellars Sea Eagles, Haliaeetus pelagicus, found dead in the northern part of Hokkaido island accumulated high levels of PCBs and DDT and metabolites. The TEQ values calculated from the PCB concentration in the eagles were high enough to cause a significant toxic effect in other birds living in the same environment. Some of these birds were also contaminated with high concentrations of lead. Spotted seals, Phoca largha, captured along the coast-line of Hokkaido accumulated PCBs in their fat at about 100 million times the concentrations in the surface sea water. The levels of expressions of hepatic microsomal CYP 1A1and related enzyme activities in these seals showed good correlation to the levels of PCBs accumulated in the fat. The fresh water crabs, Eriocheir japonicus, were captured from three different rivers with various degrees of pollution. The P450 content and the related enzyme activities showed good correlation to TEQ values obtained from the concentrations of PCBs and PCDDs in the crabs from the rivers. The wild rodents, Clethrionomys rufocanus, were captured from urban, agricultural, and forest areas in Hokkaido. Those from the forest area had the lowest CYP content and related enzyme activities, comparable to those in laboratory-raised animals. Those from the urban areas, presumably contaminated with PAHs from fuel combustion, showed increased CYP 1A1 content and related enzyme activities. Those from the agricultural areas showed increased levels of CYP 1A1, 2B, 2E1. Rats treated with some of the agrochemicals used in the area resulted in a similar pattern of induction. It is concluded that P450 can be a useful biomarker for assessing the environmental impact of chemical pollutants on wild animals.     

Glucagon-Like Peptide-1 Secretory Function as an Independent Determinant of Blood Pressure: Analysis in the Tanno-Sobetsu Study

Aims

Roles of glucagon-like peptide-1 (GLP-1) in extra-pancreatic tissues remain unclear. The aim of this study was to examine determinants of GLP-1 secretory function and possible contribution of GLP-1 to blood pressure (BP) regulation.

Methods and Results

We recruited 128 subjects who received annual examinations and 75g-oral glucose tolerance tests (OGTT) in the Tanno-Sobetsu cohort. Subjects on regular medications for cardiovascular and/or metabolic diseases were excluded, and data for the remaining 103 subjects were used for the univariate and multivariate analyses. Age, plasma glucose (PG), hemoglobin A1c (HbA1c), plasma insulin, and serum lipids were not selected as independent determinants of fasting GLP-1 level by multiple linear regression analysis. However, age and female sex were selected as independent positive determinants of the area under the curve of GLP-1 level during OGTT (AUC_GLP-1), an index of GLP-1 secretory function. Multiple linear regression analysis indicated that AUC_GLP-1 was an independent negative predictor of systolic BP (SBP), while AUC_GLP-1 was not correlated with fasting PG or HbA1c level. In subgroup analyses using the median of AUC_GLP-1 to divide the study subjects into high and low GLP-1 response groups, AUC_GLP-1 was significantly correlated with both SBP and diastolic BP (r = 0.40 and 0.28, respectively) in the low GLP-1 response group but not in the high GLP-1 response group.

Conclusions

The results of the present study suggest that GLP-1 secretory function is involved in prevention of BP elevation and that the GLP-1 response to oral glucose rather increases with aging perhaps as an adaptive phenomenon.     

A Reduction in Age-Enhanced Gluconeogenesis Extends Lifespan

The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.     

Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.