Stimulation of protein kinase C modulates insulin-like growth factor-1-induced akt activation in PC12 cells

Activation of protein kinase C (PKC) plays an important role in the negative regulation of receptor signaling, but its effect on insulin-like growth factor-1 (IGF-1) receptor signaling remains unclear. In this study, we characterized the intracellular pathways involved in IGF-1-induced activation of Akt and evaluated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on the Akt activation by IGF-1. IGF-1 induced a time- and concentration-dependent activation of Akt. The effect of IGF-1 was blocked by the phosphatidylinositide 3-kinase (PI3K) inhibitors LY294002 (50 micrometer) and wortmannin (0.5 micrometer), but not by the MEK inhibitor PD98059 (50 micrometer) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the stimulation of Akt by IGF-1 is mediated by the PI3K pathway. Interestingly, cotreatment with PMA (400 nm) attenuated IGF-1-induced activation of Akt. The attenuation was blocked completely by the PKC inhibitor GO6983 (0.5 micrometer), but only partially by the MEK inhibitor PD98059 (50 micrometer), indicating that MAPK-dependent and -independent pathways are involved. PMA induced the activation of PKC in PC12 cells, and this induction was blocked by GO6983. These data further support the role of PKC in the effect of PMA. Moreover, PKCdelta is likely involved in the action of PMA on the basis of data obtained using isoform-specific inhibitors such as rottlerin. PMA also decreased IGF-1-induced tyrosine phosphorylation of insulin receptor substrate-1 and its association with PI3K. Taken together, these results suggest, for the first time, that stimulation of PKC modulates IGF-1-induced activation of Akt.     

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoog's medium containing either N ⁶-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil. Received: 1 October 1997 / Revision received: 31 March 1998 / Accepted: 7 April 1998     

A thermoluminescence study of the effects of nitrite on photosystem II in spinach thylakoids.

The site of action of nitrite on PS II was investigated by measuring the TL profile of nitrite-treated spinach thylakoid membranes. Three bands were observed in control, which were identified as the Q band (7 degrees C), the B band (24 degrees C) and the C band (57 degrees C). In the presence of 20 mmol/L nitrite, the intensity of the Q band decreased, the B band upshifted to 46 degrees C but the C band disappeared. The suppression of the Q band and the upshift of the B band suggested that nitrite caused inhibition at the water oxidizing complex. The effects of nitrite also remained the same in the presence of chloride. In case of ion-sufficient thylakoid membranes, nitrite decreased the Q band peak intensity and caused an upshift in the B band peak temperature. Nitrite showed similar effects in the presence of DCMU. This suggested that the site of action of nitrite is not at the acceptor side but at the donor side of PS II. The inhibition shown by nitrite has been found to be specific for nitrite anion. No other anions such as formate, fluoride or nitrate, were effective.     

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

IntroductionAnnexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s).MethodsBy mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways.ResultsOur in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1.ConclusionsOur data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.     

Interaction of ATP with a Small Heat Shock Protein from Mycobacterium leprae: Effect on Its Structure and Function

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.     

Fruit and Vegetable Consumption and Cardiovascular Risk Factors in Older Chinese: The Guangzhou Biobank Cohort Study

Objective

To examine the adjusted associations of fruit consumption and vegetable consumption with the Framingham score and its components in the non-Western setting of Southern China, considering health status.

Method

Linear regression was used to assess the cross-sectional associations of fruit and vegetable consumption with the Framingham score and its components, among 19,518 older Chinese (≥50 years) from the Guangzhou Biobank Cohort Study in Southern China (2003–2006), and whether these differed by health status.

Results

The association of fruit consumption with the Framingham score varied by health status (P-value<0.001), but not vegetable consumption (P-value 0.51). Fruit consumption was associated with a lower Framingham score (-0.04 per portions/day, 95% confidence interval (CI) -0.08 to -0.004) among participants in poor health, adjusted for age, sex, recruitment phase, socio-economic position and lifestyle. However, similarly adjusted, fruit consumption was associated with a higher Framingham score (0.05, 95% CI 0.02 to 0.09) among participants in good health, perhaps due to a positive association of fruit consumption with fasting glucose. Similarly adjusted, vegetable consumption was associated with a higher Framingham score (0.03, 95% CI 0.01 to 0.05) among all participants, with no difference by health status.

Conclusion

This large study from a non-western setting found that fruit and vegetable consumption was barely associated with the Framingham score, or major CVD risk factors.     

Non-enzymatic glycation induces structural modifications of myoglobin

Increased glucose concentration in diabetes mellitus causes glycation of several proteins, leading to changes in their properties. Although glycation-induced functional modification of myoglobin is known, structural modification of the protein has not yet been reported. Here, we have studied glucose-modified structural changes of the heme protein. After in vitro glycation of metmyoglobin (Mb) by glucose at 25°C for 6 days, glycated myoglobin (GMb) and unchanged Mb have been separated by ion exchange (BioRex 70) chromatography, and their properties have been compared. Compared to Mb, GMb exhibits increased absorbance around 280 nm and enhanced fluorescence emission with excitation at 285 nm. Fluorescence quenching experiments of the proteins by acrylamide and KI indicate that more surface accessible tryptophan residues are exposed in GMb. CD spectroscopic study reveals a change in the secondary structure of GMb with decreased α-helix content. 1-anilino-naphthaline-8-sulfonate (ANS) binding with Mb and GMb indicates that glycation increases hydrophobicity of the heme protein. GMb appears to be less stable with respect to thermal denaturation and differential calorimetry experiments. Heme-globin linkage becomes weaker in GMb, as shown by spectroscopic and gel electrophoresis experiments. A correlation between glycation-induced structural and functional modifications of the heme protein has been suggested.     

Effect of metformin on renal microsomal proteins, lipid peroxidation and antioxidant status in dexamethasone-induced type-2 diabetic mice

An SDS-PAGE analysis of renal microsomal fraction of albino mice was performed to study the involvement of proteins in dexamethasone-induced type-2 diabetes mellitus (DM) and their alterations by metformin, a widely accepted oral antidiabetic drug. In addition, changes in renal lipid peroxidation (LPO), activities of superoxide dismutase (SOD) and catalase (CAT), reduced glutathione (GSH) content, as well as renal somatic index (RSI) and daily rate of water consumption were also investigated. While dexamethasone administration (1.0 mg/kg for 21 days) expressed two renal proteins (43 kDa and 63.23 kDa), in addition to the increased fasting serum levels of glucose and insulin, renal LPO, RSI and daily rate of water consumption, a parallel decrease in renal SOD, CAT and GSH was also observed. Treatment with metformin normalized these alterations including the renal proteins and LPO, confirming its efficacy in ameliorating dexamethasone-induced type-2 DM and also the association of two proteins with type-2 DM.     

Centella asiatica (L.) urban from Nepal: Quali-quantitative analysis of samples from several sites,and selection of high terpene containing populations for cultivation

Centella asiatica (L.) Urban is widely used in traditional medicine in many countries and in the formulation of drugs and cosmetics, and is therefore suitable as a trade item for the development of medicinal plants for the population of Nepal. The aim of this work was to select plant populations of C. asiatica with high contents of secondary metabolites growing in various localities in Nepal, and to enhance knowledge of the cultivation of this plant. Quali-quantitative analysis of bioactive triterpenes (asiaticoside and asiatic acid) and phenol derivatives (flavonoids and caffeoyl esters) was performed by HPLC-DAD-ELSD. The highest quantities of triterpenes and phenols were found in samples from the Gorkha and Chitwan districts. Regarding cultivated plants, soil fertilisation is critical, since over-rich soils affect secondary metabolite content. Plants growing in sand-rich soils produce more terpenes. This work provides indications on how to select high-terpene producing germplasm and recommendations for plant cultivation.