Responses of antioxidant potentials in Dioscorea rotundata Poir. following paclobutrazol drenching

The effect of paclobutrazol (PBZ) treatments on the antioxidant metabolism of white yam (Dioscorea rotundata Poir.) was investigated in the present study. PBZ @ 15 mg l(-1) plant(-1) was given to plants by soil drenching, 30, 60, and 90 days after planting (DAP). The non-enzymatic antioxidant contents like ascorbic acid (AA), reduced glutathione (GSH) and alpha-tocopherol (alpha-toc), activities of antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and catalase (CAT) were extracted and assayed on 100 DAP from leaf, stem and tubers of both control and PBZ treated plants. It was found that PBZ has a profound effect on the antioxidant metabolism and caused an enhancement in both non-enzymatic and enzymatic antioxidant potentials under treatments in white yam. Our results have good significance, as this increase the innate antioxidant potential of this food crop, which is helpful to satisfy the needs of antioxidants in diet and thereby make it an economically important food crop.     

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr)

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.     

Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1–61) of p53 via an extended binding interface, with dissociation constant of 12.7 (± 0.7) μM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1–40) and TAD2 (residues 41–61) subdomains of p53. HmtSSB enhances intrinsic 3′-5′ exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) present at 3′-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.     

Immunolocalization and serum antibody responses to Brugia malayi pepsin inhibitor homolog (Bm-33)

cDNA coding for Brugia malayi pepsin inhibitor homolog (Bm-33) from the human filarial parasite was cloned in pRSET for large-scale expression and functional characterization. The pRSET-B cloned gene did not yield recombinant protein expression and the reason was attributed to the presence of an N-terminal signal peptide. The gene was subcloned in pRSET-A without a signal peptide and the 33 kDa histidine-tagged recombinant protein was purified by IMAC. All individuals from an endemic area generated IgG responses against Bm-33 in the order MF>CP>EN. Isotype analysis indicated an elevated IgG4 reactivity in the order MF>EN>CP. Bm-33-specific IgE levels were elevated in MF, CP and EN compared to non-endemic normals with no significant differences among the groups. Paraffin-embedded sections of Setaria digitata (cattle filarial parasite) stained with mouse anti-Bm-33 antibodies exhibited the hypodermal nature of Bm-33. These findings suggest that Bm-33 is an immunodominant antigen and contributes to filarial pathogenesis.     

Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

 Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28–30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76±0.78 mg·g⁻¹ wet tissue in normal unexposed rats; 15.82±2.30 mg·g⁻¹ wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹) in comparison to normal (1.53 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase activity in order to maintain nitrogen metabolism in the initial phase of hypoxic exposure. Received: 30 March 1998 / Revised: 18 November 1998 / Accepted: 25 November 1998     

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes

N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I–P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I–P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b₆/f complex, could prevent the quenching of I–P rise by TMPD. In control thylakoids, MV suppressed the I–P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I–P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O₂ during re-oxidation of TMPD by PS I contributes towards an early decline in the I–P step of the OJIP fluorescence rise.     

Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study

2,2,2-Trifluoroethanol (TFE) is widely used to induce helix formation in peptides and proteins, but the mechanism behind this effect is still poorly understood. Several recent papers have proposed that TFE acts by selectively desolvating the peptide backbone groups of the helix state. Infrared (IR) spectroscopy of the amide I band of polypeptides can be used to probe both secondary structure and backbone solvation, making this technique well suited for addressing the effect of TFE on polypeptide conformation. In this paper, we report the IR spectra as a function of TFE concentration for an alanine-rich peptide based on the repeat (AAKAA)(n)(). The IR spectra confirm that TFE desolvates the helical state of the peptide to a greater extent than the random coil state. Moreover, using a series of specifically (13)C-labeled peptides, the precise residues desolvated in the presence of TFE were identified. The residues most desolvated by TFE are the alanines located at position i - 4 in the sequence, where i is a lysine residue. This pattern of desolvation is consistent with molecular dynamics simulations which predict strong interactions between the lysine side chain at position n and the backbone carbonyl of the alanine at position i - 4. This is the first direct spectroscopic evidence of specific desolvation of helix backbone atoms in model alanine-rich peptides.     

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.