A Simple and Non-destructive Method of Direct-PCR for Plant Systems

To date, PCR is a fundamental tool for most of the research concerning plant diversity analysis, marker-assisted selection, genetic purity testing, disease diagnostics, and transgene analysis. In all of these analyses, good-quality DNA serves as a template for amplification of target sequences. Extraction of good-quality DNA requires many steps, making the whole process time consuming, tedious, labor intensive, and expensive due to costlier and toxic chemicals. To overcome these preparatory steps from PCR-based DNA amplification, we have developed a direct-PCR amplification method for plants without isolating DNA. The method is unique and beneficial over some previously described methods of direct-PCR which fail due to inefficient amplification of target DNA in the presence of PCR inhibitors and crop specificity. Moreover, such methods are non-specific and, being destructive, cannot be replicated; one cannot completely rely on them due to lack of reproducibility. This method was streamlined from our earlier observation that alcohol-desiccated tissues maintain intact DNA for a long time. This method is specific, rapid, and, being non-destructive, allows replication, giving advantages over existing methods. The method was tested over a wide range of plant species and found very effective and quick in generating data. The method was successfully used to test the genetic purity of pearl millet hybrid (RHB-127) and its restorer (RIB 3135-18) and CMS line (ICMA 93333A). Our method is especially important for developing inexpensive and high-throughput non-invasive genetic analyses.     

Isolation and characterization of calcineurin from bovine brain

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of calmodulin and troponin-C on activities of homogeneous liver phosphoprotein phosphatases

The effect of calmodulin was determined on activities of two homogeneous liver phosphoprotein phosphatases with phosphorylase a and phosphorylated histones as substrates. Calmodulin in the absence or presence of calcium had no effect on the dephosphorylation of phosphorylase a by either phosphatases. However, calmodulin inhibited the dephosphorylation of histones catalyzed by both phosphatases. No difference was found whether the reactions were carried out in the absence or presence of calcium. The effect of calmodulin on histone dephosphorylation was variable depending on (i) the absence or presence of KCl and Mg²⁺, and (ii) the concentration of histone in the reaction mixture. In the presence of KCl and Mg²⁺ at a histone concentration of 0.1 mg/ml, calmodulin inhibited the enzyme activity. At 1 mg/ml histone, lower concentrations of calmodulin activated whereas higher concentrations of calmodulin inhibited the enzyme activity. Similar, but relatively less, effect was observed with troponin-C. In the absence of KCl and Mg²⁺, calmodulin as well as troponin-C activated the enzyme activity. The optimal concentration of calmodulin (or troponin-C) was approximately 30–50% of histone concentration in the reaction mixture. Calcium alone or with calmodulin (or troponin-C) had no additional effect on enzyme activities. DNA and RNA, two negatively charged nucleic acids, also showed similar effects on histone dephosphorylation. Depending on whether KCl and Mg²⁺ were absent or present in the reaction mixtures, both nucleic acids either activated or inhibited the dephosphorylation of histones.     

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70 in HepG2 and HepG2 cells overexpressing constitutively active Akt/PKB

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70^S6K). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70^S6K (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70^S6K in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70^S6K in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r² = 0.525, p < 0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.     

Intrinsic phosphatase activity of bovine brain calcineurin requires a tightly bound trace metal

The divalent metal requirement of intrinsic phosphatase activity was investigated using native and trypsinized calcineurin. This was assessed by examining (1) the stimulation of the enzyme by various metals, (2) the inhibition of the enzyme activity by metal chelators (EDTA and EGTA), and (3) the restoration by various metals of the activity of the EDTA-inhibited calcineurin phosphatase. The results supported the view that a tightly bound trace metal is necessary for expression of the phosphatase activity of calcineurin and implicate Mn2+ as the tightly bound metal.     

Glycogen metabolizing enzyme activities in the developing rat liver

1. The glycogen content of rat liver increased prenatally and dramatically decreased after the birth. 2. Glycogen synthase and glycogen phosphorylase activities increased prenatally, declined at 12 hr after birth and then again increased. 3. Phosphorylase kinase activities did not significantly change prenatally but steadily increased after the birth. 4. Protein kinase activities (units/mg liver) did not change prenatally but slightly increased after the birth. 5. Phosphoprotein phosphatase activities using phosphorylase a and histone as substrates dramatically increased at birth and then decreased after 24 hr of the birth.