Induction of chitinase activity by exogenous cytokinins in excised dark-grown cucumber cotyledons: involvement of Ca and staurosporine-sensitive protein kinase(s) in cytokinin signaling

Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal CaCl₂ and calcium ionophore increased the basal chitinase activity by 6- and 5-fold, respectively. Moreover, the effects of staurosporine, sodium fluoride, and Ca²⁺ on Z-induced chitinase activity correlate with their effects on chitinase protein levels. Taken together, our data suggests Ca²⁺ and staurosporine-sensitive protein kinase(s) as components of the cytokinin transduction machinery involving induction of chitinase in cucumber.     

Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²⁺ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K_m values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k_cat value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.     

Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice

Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2–4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent with in vitro studies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP''s protective effects in the lung.     

Evaluation of Curcumin Capped Copper Nanoparticles as Possible Inhibitors of Human Breast Cancer Cells and Angiogenesis: a Comparative Study with Native Curcumin

Synthesis of metal nanoparticles for improving therapeutic index and drug delivery is coming up as an attractive strategy in the mainstream of cancer therapeutic research. In the present study, curcumin-capped copper nanoparticles (CU-NPs) were evaluated as possible inhibitors of in vivo angiogenesis, pro-angiogenic cytokines involved in promoting tumor angiogenesis along with inhibition of cell proliferation and migration of breast cancer cell line MDA-MB-231. The antiangiogenic potential was assessed using in vivo chorioallantoic membrane (CAM) model. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based cytotoxicity assay was used to assess the effect of CU-NPs against proliferation of breast cancer cell line. The wound healing migration assay was used to evaluate the effects of CU-NPs on the migration ability of breast cancer cell line. Native curcumin (CU) was used as a reference compound for comparison purpose. The result of the present investigation indicates that CU-NPs could not demonstrate impressive antiangiogenic or anticancer activities significantly as compared to native CU. The possible mechanisms of experimental outcomes are discussed in the light of the methods of nanoparticle synthesis in concert with the current state of the art literature.     

Poor Long-Term Efficacy of Prevnar-13 in Sickle Cell Disease Mice Is Associated with an Inability to Sustain Pneumococcal-Specific Antibody Titers

Background

One of the most common causes of morbidity and mortality in children with sickle cell disease (SCD) is infection with the pneumococcal bacterium (Streptococcus pneumoniae). Unfortunately, the polysaccharide-conjugate vaccine appears to be less effective in individuals with SCD when compared to the general population. We sought to better understand the relative efficacy of pneumococcal vaccination in a SCD mouse challenge model.

Methods

Transgenic control and SCD mice were monitored for mortality after intranasal pneumococcal infection or pneumococcal vaccination with Prevnar-13 and type-matched challenge. Anti-pneumococcal antibody titers were measured by ELISA and opsonophagocytosis was measured in vitro.

Results

Mortality after pneumococcal infection was similar between control and SCD mice. However, after three intramuscular polysaccharide-conjugate vaccinations, all control mice were protected following high-dose intranasal infection, whereas 60% of SCD mice died. Anti-pneumococcal antibody titers showed initial IgG and IgM responses in both groups, but waning titers were observed in the SCD group, even after boosting. When functionally assayed in vitro, serum from SCD mice 13 weeks after a second booster shot maintained little to no ability to opsonize pneumococci, while serum from control mice sustained a significantly higher capacity opsonization. Thus, it appears that SCD mice do not maintain antibody responses to pneumococcal polysaccharides after Prevnar-13 vaccination, thereby leaving them susceptible to mortality after type-matched infection.

Conclusion

Our results emphasize the need to better understand the correlates of immune protection in SCD so that pneumococcal vaccines can be improved and mortality reduced in this susceptible population.     

Nutrient utilisation,growth performance and blood metabolites in Murrah buffalo calves (Bubalus bubalis) divergently selected for residual feed intake

The aim of this study was to evaluate differences in efficiency of feed utilisation between buffalo calves with low and high residual feed intake (RFI) by comparing feed intake, nutrient digestibility, growth traits and blood metabolites. Eighteen male Murrah buffalo calves (aged 4–6 months; 70 ± 1.0 kg body weight) were fed ad libitum with a total mixed ration for 120 d. Based on linear regression models involving dry matter intake (DMI), average daily gain (ADG) and mid-test metabolic body size, calves were assigned into low and high RFI groups. The RFI varied from ?0.33 to +0.28 kg DM/d with an average RFI of ?0.14 and 0.14 kg DM/d in low and high RFI calves, respectively. Calves had a mean DMI of 1.9 and 2.4 kg/d and an ADG of 0.5 and 0.6 kg/d in low and high RFI groups, respectively. Low RFI calves ate 19.0% less DM each day and required significantly less metabolisable energy for maintenance compared with high RFI calves (12.5 vs. 16.7 MJ/d). Nutrient digestibility and nitrogen balance did not differ among low and high RFI calves. In more efficient animals (low RFI calves) higher (p < 0.05) plasma level of growth hormone, insulin-like growth factor-1 (IGF-1), triiodothyronine (T₃) and lower concentration of thyroxin hormone were detected. No significant differences in levels of insulin, hydroxyproline, plasma and urine creatinine, total protein and albumin between high and low RFI groups were found. Blood metabolites showed significant (p < 0.05) differences at initial and final stages of study in both groups. At final stage of study, RFI showed negative correlations with growth hormone, IGF-1, T₃, urine creatinine and albumin. Low RFI buffalo calves are more efficient in feed utilisation and the differences in blood metabolites are probably due to differences in feed intake and body metabolism.     

Frankixalus,a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic ‘Polypedates’ jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.     

MicroRNA mediated regulation of gene expression in response to heavy metals in plants

Journal of Plant Biochemistry and Biotechnology - Plants being sessile organisms are exposed to innumerable abiotic and biotic stresses on a daily basis. Heavy metal toxicity in plants results in...