Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

Identification of a CYP84 family of cytochrome P450-dependent mono-oxygenase genes in Brassica napus and perturbation of their expression for engineering sinapine reduction in the seeds

CYP84 is a recently identified family of cytochrome P450-dependent mono-oxygenases defined by a putative ferulate-5-hydroxylase (F5H) from Arabidopsis. Until recently F5H has been thought to catalyze the hydroxylation of ferulate to 5-OH ferulate en route to sinapic acid. Sinapine, a sinapate-derived ester in the seeds, is antinutritional and a target for elimination in canola meal. We have isolated three F5H-like genes (BNF5H1-3) from a cultivated Brassica napus, whose amphidiploid progenitor is considered to have arisen from a fusion of the diploids Brassica rapa and Brassica oleracea. Two cultivated varieties of the diploids were also found to contain BNF5H3 and additionally either BNF5H1 or BNF5H2, respectively. Whereas all three are >90% identical in their coding sequence, BNF5H1 and BNF5H2 are closer to each other than to BNF5H3. This and additional data suggest that the two groups of genes have diverged in an ancestor of the diploids. B. napus showed maximal F5H expression in the stems, least in the seeds, and subtle differences among the expression profiles of the three genes elsewhere. Transgenic B. napus with cauliflower mosaic virus 35S-antisense BNF5H contained up to 40% less sinapine, from 9.0 +/- 0.3 mg in the controls to 5.3 +/- 0.3 mg g(-1) seed. F5H from Arabidopsis and a similar enzyme from sweetgum (Liquidamber styraciflua) has recently been shown to have coniferaldehyde hydroxylase activity instead of F5H activity. Thus the supply of 5-OH coniferaldehyde or 5-OH ferulate has a bearing on sinapine accumulation in canola seeds.     

Hesperidin, a flavanone glycoside, on lipid peroxidation and antioxidant status in experimental myocardial ischemic rats

Myocardial infarction continues to be a leading cause of mortality world-wide. Novel therapies are needed to treat the myocardial ischemia. This study was undertaken to evaluate the cardioprotective role of hesperidin on isoproterenol-induced myocardial ischemia in rats. Myocardial ischemia was induced by subcutaneous injection of isoproterenol hydrochloride (85 mg/kg body weight), for two consecutive days. Isoproterenol-administered rats showed elevated levels of cardiac markers (aspartate transaminase, alanine transaminase, lactate dehydrogenase, creatine kinase, creatine kinase-MB, cardiac troponins T and I) when compared with control and hesperidin treatment groups (100, 200 and 400 mg/kg body weight). The serum levels of cardiac markers were significantly reduced at the doses of 200 mg and 400 mg. All further experiments were carried out at the 200 mg dose. Lipid peroxidation markers (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes) were elevated significantly in the plasma and heart whereas non-enzymic antioxidants (vitamin C, vitamin E and reduced glutathione) were decreased significantly. Activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase declined significantly in the heart of ischemic rats. However, after hesperidin treatment, all the above parameters reverted to normal levels. This study demonstrated that the cardioprotective effect of hesperidin on ischemic rats could be due to its anti-lipid peroxidative and antioxidant properties.     

Molecular docking analysis of furfural and isoginkgetin with heme oxygenase I and PPARγ

It is of interest to document the molecular docking analysis based binding data of furfural and isoginkgetin with heme oxygenase I and PPARγ in the context of inflammation for further consideration in drug design and development.     

Antifungal Potential of Plant Growth Promoting Bacillus Species Against Blossom Blight of Rose

Blossom blight caused by Botrytis cinerea is one among the most devastating diseases that cause complete post-harvest loss in flower crops. The present study focuses on the development of effective bioformulation towards suppression of blossom blight and plant growth promotion in rose. Bacillus amyloliquefaciens (VB2) and Bacillus subtilis (AP) effectively inhibited mycelial growth of B. cinerea in vitro. Genome screening of VB2 and AP revealed the presence of antimicrobial peptide genes including, ituD, ipa14, bacA, bacD, srfA, sfP, spaC, spaS responsible for the biosynthesis of antibiotics such as iturin, bacilysin, bacillomycin, surfactin and subtilin. Further, the presence of volatile antifungal compounds in the bacterial secretome was identified through gas chromatography–mass spectrometry (GC/MS) analysis. Upon treatment, AP accelerated the metabolite profile of the plants and a rise in peak area of antifungal compounds such as, pentadecanoic acid, n-hexadecanoic acid, octadecanoic acid (stearic acid) and tetradecanoic acid was observed. In vitro, VB2 produced maximum indole acetic acid (9.17 µg/ml) and gibberellic acid (8.20 µg/ml) in nutrient broth. Under field conditions, foliar spray of VB2 at 0.5% (5 ml/l), four times at weekly interval suppressed blossom blight incidence (64% reduction over control) and also promoted yield. Future research towards development of an effective bioformulation with extended shelf life will aid in the management of various fungal, bacterial and viral diseases in different crop plants.

     

Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.     

Relationships between antigen-specific helper and inducer suppressor T cell hybridomas

Ts1, or inducer suppressor T cells, share many phenotypic and functional characteristics with helper/inducer subset of T cells. In order to evaluate the relationship between these cell types, we made a series of new Ts1 hybridomas by the fusion of Ts1 cells with the functionally TCR alpha/beta-negative BW thymoma (BW 1100). Three Ts1 hybridomas (CKB-Ts1-38, CKB-Ts1-53, and CKB-Ts1-81) were established that express TCR and produce Ag-specific suppressor factors constitutively, thus making it possible to study the nature and specificity of Ag receptors, MHC restriction, and lymphokine production by the Ts1 hybridomas. Results presented in this report demonstrate that all the Ts1 hybridomas described here express CD3-associated TCR-alpha beta. These three Ts1 hybridomas recognize Ag (NP-KLH) specifically in a growth inhibition assay and this recognition is restricted by IE molecules. Two of the hybridomas also produce IL-2 or IL-2 and IL-4 upon Ag-specific activation. Thus, by these three criteria the Ts1 hybridomas appear indistinguishable from Th cells. These three Ts1 hybridomas, however, release suppressor factors (TsF1) in the supernatant that suppress both in vivo DTH and in vitro PFC responses in an Ag-specific manner. Like the TsF1 factors characterized previously, the suppression mediated by these factors are Igh restricted and lack H-2 restriction. These factors mediate suppression when given in the induction phase but not during the effector phase of the immune response. The TsF1 factors are absorbed by Ag (NP-BSA), and anti-TCR affinity columns and the suppressor activity can be recovered by elution. The data are consistent with the interpretation that Ts1 inducer-suppressor T cells are related to Th cells; the feature that distinguishes these cells is the ability to produce Ag-binding factors that specifically suppress immune responses.     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478