Hydrodynamic-based delivery of an interleukin-22-Ig fusion gene ameliorates experimental autoimmune myocarditis in rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

Quantitative detection of asbestos fiber in gravelly sand using elastic body-exposure method

Chrysotile or crocidolite colloidal solution containing donor plasmid DNA and Escherichia coli cells was subjected to elastic body friction. These acicular clay minerals mediated E. coli antibiotic resistance plasmid transformation. Other clay minerals had no effect on E. coli transformation. The number of E. coli transformants was counted after elastic body exposure with various crocidolite concentrations. There was a correlation between the number of E. coli transformants and crocidolite concentration (between 40 and 1,000 ng/ml). A mixture consisting of sea sand and crocidolite was utilized as a model for quantitative detection of asbestos in gravelly sand. With sea sand containing 0.15–15 mg of crocidolite, a correlation between crocidolite concentration and the number of colonies derived from E. coli transformants was observed. This indicates that measurement of asbestos is possible even when the asbestos sample includes gravelly sand. Fluorescence microscopic observation of crocidolite colloidal solution indicated that crocidolite was present as spherical aggregates having diameters of 6–9 μm. Thus, the number of transformants correlated with that of 6–9 μm crocidolite aggregates.     

Phototrophic growth of a Rubisco-deficient mesophilic purple nonsulfur bacterium harboring a Type III Rubisco from a hyperthermophilic archaeon

The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc(Tk)). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc(Tk), here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO2 fixation. Via double-crossover homologous recombination, we first deleted three Rubisco genes present on the chromosome of the photosynthetic mesophile Rhodopseudomonas palustris No. 7. The mutant strain (delta3) could neither grow under photoautotrophic nor photoheterotrophic conditions. We introduced the rbc(Tk) gene into strain delta3 either on a plasmid, or by integrating the gene onto the chromosome. The two transformant strains harboring rbc(Tk) displayed growth under photoautotrophic and photoheterotrophic conditions, both dependent on CO2 fixation. Specific growth rates and Rubisco activity levels were compared under photoheterotrophic conditions among the two transformants and the wild-type strain. We observed that the levels of Rubisco activity in the respective cell-free extracts correlated well with the specific growth rates. Immunoprecipitation experiments revealed that Rubisco activity detected in the transformants was derived solely from Rbc(Tk). These results demonstrated that the Type III Rbc(Tk) from a hyperthermophile could support CO2 fixation in a mesophilic organism, and that the specific growth rate of the transformant can be used as a convenient parameter for selection of engineered proteins with improved Rubisco activity.     

Convenient treatment of acetonitrile-containing wastes using the tandem combination of nitrile hydratase and amidase-producing microorganisms

This study aimed to construct an acetonitrile-containing waste treatment process by using nitrile-degrading microorganisms. To degrade high concentrations of acetonitrile, the microorganisms were newly acquired from soil and water samples. Although no nitrilase-producing microorganisms were found to be capable of degrading high concentrations of acetonitrile, the resting cells of Rhodococcus pyridinivorans S85-2 containing nitrile hydratase could degrade acetonitrile at concentrations as high as 6 M. In addition, an amidase-producing bacterium, Brevundimonas diminuta AM10-C-1, of which the resting cells degraded 6 M acetamide, was isolated. The combination of R. pyridinivorans S85-2 and B. diminuta AM10-C-1 was tested for the conversion of acetonitrile into acetic acid. The resting cells of B. diminuta AM10-C-1 were added after the first conversion involving R. pyridinivorans S85-2. Through this tandem process, 6 M acetonitrile was converted to acetic acid at a conversion rate of >90% in 10 h. This concise procedure will be suitable for practical use in the treatment of acetonitrile-containing wastes on-site.     

Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2

We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO₃ in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO₃, respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.     

Sex difference in body weight gain and leptin signaling in hypocretin/orexin deficient mouse models

Recent studies in human and animal models of narcolepsy have suggested that obesity in narcolepsy may be due to deficiency of hypocretin signaling, and is also under the influence of environmental factors and the genetic background. In the current study, using two hypocretin/orexin deficient narcoleptic mouse models (i.e. preproorexin knockout (KO) and orexin/ataxin-3 transgenic (TG) mice) with cross-sectional assessments, we have further analyzed factors affecting obesity. We found that both KO and TG narcoleptic mice with mixed genetic backgrounds (N4-5, 93.75-96.88% genetic composition of C57BL/6) tended to be heavier than wild type (WT) mice of 100-200 days old. The body weight of heterozygous mice was intermediate between those of KO and WT mice. Obesity was more prominent in females in both KO and TG narcoleptic mice and was associated with higher serum leptin levels, suggesting a partial leptin resistance. Obesity is less prominent in the congenic TG narcoleptic mice, but is still evident in females. Our results confirmed that hypocretin/orexin ligand deficiency is one of the critical factors for the obese tendency in narcolepsy. However, multiple factors are also likely to affect this phenotype, and a sex difference specific alteration of leptin-hypocretin signaling may be involved.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.