Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreatic beta cell lines

Current therapy for type 1 diabetes mellitus involves a daily regimen of multiple subcutaneous or intramuscular injections of recombinant human insulin. To achieve long-term insulin delivery in vivo, we investigated the applicability of cytomedical therapy using beta TC6 cells or MIN6 cells, both of which are murine pancreatic beta cell lines that secrete insulin in a subphysiologically or physiologically regulated manner, respectively. We examined this therapy in the insulinopenic diabetic mice intraperitoneally injected with beta TC6 cells or MIN6 cells microencapsulated within alginate-poly(L)lysine-alginate membranes (APA-beta TC6 cells or APA-MIN6 cells). The diabetic mice treated with APA-beta TC6 cells fell into hypoglycemia, whereas those injected with APA-MIN6 cells maintained normal blood glucose concentrations for over 2 months without developing hypoglycemia. In addition, we also conducted an oral glucose tolerance test using these mice. The blood glucose concentrations of normal and of diabetic mice injected with APA-MIN6 cells similarly changed over time, although the blood insulin concentration increased later in the injected diabetic mice than in the former. These results suggest that cytomedicine utilizing microencapsulated pancreatic beta cell lines with a physiological glucose sensor may be a beneficial and safe therapy with which to treat diabetes mellitus.     

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

Effects of pravastatin sodium on mevalonate metabolism in common marmosets

In experimental animals and humans, the concentration of serum mevalonate (MVA), a direct product of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is considered to reflect the activity of whole-body sterol synthesis. The relationship between the concentration of serum MVA and the activity of sterol synthesis in tissues, however, has not been fully clarified. In the present study, we examined MVA metabolism by using pravastatin, a liver-selective inhibitor of HMG-CoA reductase, and common marmosets, a good model animal for studying lipid metabolism. In the time course study, the maximal reduction in the concentration of serum MVA was observed 2 h after a single oral administration of 30 mg/kg pravastatin to common marmosets. We, therefore, examined the relationship between the concentrations of serum and hepatic MVA, and sterol synthesis in some tissues at this time point. Sterol synthesis was determined ex vivo in tissue slices by measuring the incorporation of [14C]acetate into digitonin-precipitable [14C]sterols. Pravastatin at 0.03-30 mg/kg reduced dose-dependently the activity of hepatic sterol synthesis, whereas no significant reduction of sterol synthesis was observed in other tissues such as intestine, kidney, testis and spleen, even with the highest dose (30 mg/kg). The liver-specific inhibition of sterol synthesis caused parallel reductions in the concentrations of both serum and liver MVA. In addition, there were good correlations between the concentration of either serum or hepatic MVA and the activity of hepatic sterol synthesis. These data indicate that the major origin of serum MVA is the liver, and that the concentration of serum MVA reflects the concentration of hepatic MVA and the activity of hepatic sterol synthesis 2 h after a single oral administration of pravastatin in common marmosets.     

The effect of sodium acetate on the growth yield,the production of L- and D-lactic acid,and the activity of some enzymes of the glycolytic pathway of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T)

The effect of sodium acetate was studied on the change of the growth yield, the production of L- and D-lactic acid, and the activity of lactate dehydrogenases (LDHs; L-lactate dehydrogenase [EC 1.1.1.27, L-LDH] plus D-lactate dehydrogenase [EC 1.1.1.28, D-LDH]), fructose-1, 6-bisphosphate aldolase [EC 4.1.2.13, FBP-aldolase], and phosphofructokinase [EC 2.7.1.11, PFK] of Lactobacillus sakei NRIC 1071(T) and Lactobacillus plantarum NRIC 1067(T). The growth yield of L. sakei NRIC 1071(T) was increased 1.6 times in the presence of sodium acetate compared with its absence. The activity of LDHs in L. sakei NRIC 1071(T) and L. plantarum NRIC 1067(T) was retained longer under the addition of sodium acetate in the reaction mixture. As a result, these strains produced much more lactic acid in the presence of sodium acetate compared with its absence. Furthermore, the activity of L-LDH in L. sakei NRIC 1071(T) cultivated in the presence of sodium acetate increased three times or more compared with the activity of the cells cultivated in its absence. Consequently, the type of stereoisomers of lactic acid produced by L. sakei shifted from the DL-type to the L-type because the ratio of L-lactic acid to D-lactic acid produced became larger with the addition of sodium acetate to culture media. This phenomenon was not observed in L. plantarum NRIC 1067(T). Further, the participation of lactate racemase is discussed from the viewpoint of the production of D-lactic acid by L. sakei.     

High-level expression of naked DNA delivered to rat liver via tail vein injection

Background

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Methods

We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.

Results

We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.

Conclusions

These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.

     

Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.     

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.