Combination of dipeptidylpeptidase IV inhibitor and low dose thiazolidinedione: preclinical efficacy and safety in db/db mice

Thiazolidinediones (TZDs) are currently the most efficacious class of oral antidiabetics. However, they carry the burden of weight gain and haemodilution, which may lead to cardiovascular complications. The present study was designed to ascertain whether a combination of dipeptidyl peptidase IV (DPP IV) inhibitor with low dose of a thiazolidinedione absolves TZD associated weight gain and oedema without compromising its efficacy. In this study, we examined the efficacy and safety of lower dose (1 mg/kg/day) of rosiglitazone, a thiazolidinedione, in combination with 5 mg/kg/day dose of LAF-237 (vildagliptin), a known DPP IV inhibitor, in aged db/db mice after 14 days of treatment and compared the combination with therapeutic dose (10 mg/kg) of rosiglitazone. The combination therapy showed similar efficacy as that of 10 mg/kg/day rosiglitazone in lowering random blood glucose (53.8%, p<0.001 and 54.3%, p<0.001 respectively), AUC ((0-120) min) during oral glucose tolerance test (OGTT) (38.6 %, p<0.01; 38.3%, p<0.01 respectively) and triglyceride levels (63.9% and 61% respectively; p<0.01). Plasma active glucagon like peptide-1 (GLP-1) and insulin levels were found to be elevated significantly (p<0.01 and p<0.05 respectively) in both LAF-237 and combination treated groups following oral glucose load. LAF-237 alone had no effect on random glucose and glucose excursion during OGTT in severely diabetic db/db mice. Interestingly, the combination treatment showed no significant increase in body weight as compared to the robust weight gain by therapeutic dose of rosiglitazone. Rosiglitazone at 10 mg/kg/day showed significant reduction (p<0.05) in haematocrit, RBC count, haemoglobin pointing towards haemodilution associated with increased mRNA expression of Na(+), K(+)-ATPase-alpha and epithelial sodium channel gamma (ENaCgamma) in kidney. The combination therapy escaped these adverse effects. The results suggest that combination of DPP IV inhibitor with low dose of thiazolidinedione can interact synergistically to represent a therapeutic advantage for the clinical treatment of type 2 diabetes without the adverse effects of haemodilution and weight gain associated with thiazolidinediones.     

Development of a water hyacinth based vermireactor using an epigeic earthworm Eisenia foetida

The aim of this work was to investigate the potential of water hyacinth (WH) spiked with cow dung (CD) into vermicompost. Five vermireactors containing WH and CD in different ratios, were run under laboratory conditions for 147 days. The maximum worm growth was recorded in CD alone. Worms grew and reproduced favourably in 25% WH+75% CD feed mixture. Greater proportion of WH in feed mixture significantly affected the biomass gain, hatchling numbers and numbers of cocoons produced during experiments. In all the vermireactors, there was significant decrease in pH, TOC and C:N ratio, but increase in TKN, TK and TAP at the end. The heavy metals content in the vermicomposts was lower than initial feed mixtures. The results indicated that WH could be potentially useful as raw substrate in vermicomposting if mixed with up to 25% in cow dung (on dry weight basis).     

Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7

Ten different strains of Thermomyces lanuginosus, isolated from composting soils were found to produce phytase when grown on PSM medium. The wild type strain CM was found to produce maximum amount ofphytase (4.33 units/g DW substrate). Culturing T. lanuginosus strain CM on medium containing wheat bran and optimizing other culture conditions (carbon source, media type, nitrogen source, level of nitrogen, temperature, pH, inoculum age, inoculum level and moisture), increased the phytase yield to 13.26 units/g substrate. This culture was further subjected to UV mutagenesis for developing phytase hyperproducing mutants. The mutant (TL-7) showed 2.29-fold increase in phytase activity as compared to the parental strain. Employing Box-Behnken factor factorial design of response surface methodology resulted in optimized phytase production (32.19 units/g of substrate) by mutant TL-7. A simple two-step purification (40.75-folds) ofphytase from mutant TL-7 was achieved by anion exchange and gel filtration chromatography. The purified phytase (approximately 54 kDa) was characterized to be optimally active at pH 5.0 and temperature 70 degrees C, though the enzyme showed approximately 70% activity over a wide pH and temperature range (2.0-10.0 and 30-90 degrees C, respectively). The phytase showed broad substrate specificity with activity against sodium phytate, ADP and riboflavin phosphate. The phytase from T. lanuginosus was thermoacidstable as it showed up to 70% residual activity after exposure to 70 degrees C at pH 3.0 for 120 min. The enzyme showed Km 4.55 microM and Vmax 0.833 microM/min/mg against sodium phytate as substrate.     

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T₁ lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T₁ plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.     

Adaptation of a Silica Membrane-based Kit for Purification of Sequencing-ready BAC DNA

We describe a modification of a protocol for the isolation of BAC DNA using a silica membrane-based kit designed for the isolation of plasmid DNA. The major advantages of this protocol are the expediency of the procedure, the high yield and purity, and the high quality of the BAC DNA that is suitable for direct sequencing.     

Prognostic significance of the histomorphometric features of bone marrow trephine biopsies in patients with chronic myeloid leukemia

OBJECTIVE: To study the correlation of histomorphometric data of bone marrow trephine biopsies at the time of initial diagnosis in chronic myeloid leukemia (CML) patients with the patient prognosis. STUDY DESIGN: A total of 40 CML patients were divided equally in group I (developed accelerated phase or blast crisis within 18 months of initial diagnosis of chronic phase of CML) and group II (developed accelerated phase or blast crisis > 30 months after initial diagnosis of chronic phase of CML). The clinical, hematologic and histomorphometric data were compared in the 2 groups of CML patients. RESULTS: The percentage of bone marrow promyelocytes was significantly increased in group I. On morphometry, the number of blasts per square millimeter, the area of reticulin fibers per square millimeter and the percentage area occupied by reticulin fibers were statistically significant. CONCLUSION: There seems to be grounds for hope for predicting prognosis of CML patients at initial diagnosis based on histomorphometric findings. The percentage area of reticulin fibers and the number of blasts per square millimeter are important prognostic predictors in histomorphometry data.     

The natural killer cell dysfunction of aged mice is due to the bone marrow stroma and is not restored by IL‐15/IL‐15Rα treatment

Immune dysfunctions in the elderly result in increased susceptibility to infectious diseases, cancer, and autoimmune diseases. Natural killer (NK) cells are bone marrow‐derived lymphocytes crucial for host defense against several infections and cancer. We have previously shown that compared to young, aged C57BL/6 mice have decreased numbers of mature NK cells in the blood, spleen, and bone marrow, resulting in susceptibility to mousepox, a lethal disease caused by ectromelia virus. Here, we describe further age‐related defects in NK cells including reduced proliferation in vivo, additional signs of immaturity, and dysregulated expression of activating and inhibitory receptors. Aging also alters the expression of collagen‐binding integrins in conventional NK cells and the frequency and phenotype of liver tissue‐resident NK cells. We additionally show that the defect in NK maturation is the consequence of deficient maturational cues provided by bone marrow stromal cells. Moreover, we demonstrate that in aged mice, treatment with complexes of the cytokine IL‐15 and IL‐15Rα induce massive expansion of the NK cells, but most of these NK cells remain immature and are unable to restore resistance to mousepox. The use of rodent model to understand immunosenescence may help the development of treatments to improve the immune fitness of the aged. Our work with NK cells should contribute toward this goal.     

Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit

Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.