Elevated immunoreactive-somatostatin levels in the brain of ataxic mutant mice

Immunoreactive-somatostatin (IR-SRIF) levels were investigated in the brain of 4 types of ataxic mice (Rolling Mouse Nagoya, Weaver, PCD, Staggerer) with different cerebellar pathologies. IR-SRIF concentrations (ng/mg) were found to be significantly elevated in both cerebellum and cerebrum of all ataxic mutant mice, IR-SRIF (ng/organ) was found to be increased in the cerebellum and cerebrum in Rolling Mouse Nagoya and PCD compared with control mice. The gel-filtration profile (Sephadex G-50) in the cerebellar extracts of Rolling Mouse Nagoya proved to be identical to that of control mice. Three peaks of IR-SRIF were found to be uniformly elevated in Rolling Mouse Nagoya, with the highest peak coinciding with authentic somatostatin-14. The present results suggest that elevated levels of IR-SRIF in the brain may play a role in the mechanism underlying the manifestation of ataxia in ataxic mutant mice, especially in Rolling Mouse Nagoya and PCD.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

Nifedipine, a calcium channel blocker, inhibits inflammatory and fibrogenic gene expressions in advanced glycation end product (AGE)-exposed fibroblasts via mineralocorticoid receptor antagonistic activity

Advanced glycation end products (AGE) are involved in tissue damage and remodeling. This study investigated whether AGE could elicit inflammatory and fibrogenic reactions in fibroblast cell line MRC-5 cells via autocrine production of aldosterone and if nifedipine could block the AGE actions through mineralocorticoid receptor (MR) antagonistic activity. AGE significantly up-regulated monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β (TGF-β), type III collagen and receptor for AGE (RAGE) mRNA levels in MRC-5 cells, all of which were completely blocked by nifedipine or an MR antagonist spironolactone. Aldosterone also dose-dependently increased MCP-1, TGF-β and type III collagen mRNA levels in MRC-5 cells, which were suppressed by nifedipine, but not amlodipine, a control calcium channel blocker. Further, AGE significantly stimulated aldosterone generation in MRC-5 cells, which was partially blocked by nifedipine or spironolactone. In this study, we demonstrated for the first time that AGE could evoke inflammatory and fibrogenic reactions in MRC-5 cells via aldosterone production, which were blocked by the MR antagonistic activity of nifedipine. Our present study provides a unique beneficial aspect of nifedipine on tissue damage and remodeling; it could work as an anti-inflammatory and anti-fibrogenic agent against AGE via MR antagonistic activity.     

Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.     

Antidiabetic effect of Lactobacillus GG in streptozotocin-induced diabetic rats

Neonatally streptozotocin-induced diabetic (n-STZ) rats were given food containing Lactobacillus GG cells (GG) or a control diet (control), from 9 to 18 weeks of age. The GG cells significantly lowered the blood hemoglobin A(1C) (HbA(1C)) level and improved glucose tolerance in n-STZ rats (p<0.05). In the GG group, the serum insulin level at 30 min after glucose loading was significantly higher than in the control group (p<0.05).     

Peptidoglycan‐induced T helper 2 immune response in mice involves interleukin‐10 secretion from Langerhans cells

Patients with atopic dermatitis (AD) have superficial skin colonization with Staphylococcus aureus and an increased number of T helper (Th)2 cells in their peripheral blood. The purpose of this study was to clarify the involvement of interleukin (IL)‐10 secretion from Langerhans cells (LCs) in staphylococcal peptidoglycan (PEG)‐induced Th2 immune responses in mice. Mice were primed with LCs pulsed with PEG (or LPS) and ovalbumin (OVA) and then given a booster OVA injection 2 days later in the hind footpad. Five days after the OVA injection, cytokine responses in the draining popliteal lymph nodes were investigated by RT‐PCR and ELISA. Production of both IL‐10 and IL‐12 by cultured LCs was detected by ELISA. Administration of PEG‐ or LPS‐stimulated LCs into the hind footpads of the mice induced Th2‐prone and Th1‐prone immune responses, respectively, as represented by expression of IL‐4 and interferon ‐γ . In vitro experiments showed that PEG induced greater production of IL‐12 p40 from LCs than did LPS, whereas LPS induced greater production of IL‐12 p70 from LCs than did PEG. Furthermore, it was found that PEG‐stimulated LCs induced greater production of IL‐10 than did LPS‐stimulated LCs, and that neutralization of IL‐10 augmented IL‐12 p70 production and inhibited Th2 development by PEG‐stimulated LCs. These results suggest that PEG can induce Th2 development through down‐regulation of IL‐12 p70 production by LCs in an IL‐10 production‐dependent manner and would explain the role of S. aureus colonization in patients with AD.     

Molecular and morphological evidence support four new species in the genus Muscodor from northern Thailand

The genus Muscodor comprises fungal endophytes which produce mixtures of volatile compounds (VOCs) with antimicrobial activities. In the present study, four novel species, Muscodor musae, M. oryzae, M. suthepensis and M. equiseti were isolated from Musa acuminata, Oryza rufipogon, Cinnamomum bejolghota and Equisetum debile, respectively; these are medicinal plants of northern Thailand. The new Muscodor species are distinguished based on morphological and physiological characteristics and on molecular analysis of ITS-rDNA. Volatile compound analysis showed that 2-methylpropanoic acid was the main VOCs produced by M. musae, M. suthepensis and M. equiseti. The mixed volatiles from each fungus showed in vitro antimicrobial activity. Muscodor suthepensis had the highest antifungal activity.     

Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.     

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T = 3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~ 350 Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~ 440 Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. &; Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA 108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.