Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties

Several decades back ascorbic acid was proposed as an effective anticancer agent. However, this idea remained controversial and the mechanism of action unclear. In this paper, we show that ascorbic acid at a concentration reported to be achievable through high doses of oral consumption is capable of cytotoxic action against normal cells. Several antioxidants of both animal as well as plant origin including ascorbic acid also possess prooxidant properties. Copper is an essential component of chromatin and can take part in redox reactions. Previously we have proposed a mechanism for the cytotoxic action of plant antioxidants against cancer cells that involves mobilization of endogenous copper ions and the consequent generation of reactive oxygen species. Using human peripheral lymphocytes and Comet assay we show here that ascorbic acid is able to cause oxidatative DNA breakage in normal cells at a concentration of 100–200 μM. Neocuproine, a Cu(I) specific sequestering agent inhibited DNA breakage in a dose dependent manner indicating that Cu(I) is an intermediate in the DNA cleavage reaction. The results are in support of our above hypothesis that involves events that lead to a prooxidant action by antioxidants. The results would support the idea that even a plasma concentration of around 200 μM would be sufficient to cause pharmacological tumor cell death particularly when copper levels are elevated. This would account for the observation of several decades back by Pauling and co-workers where oral doses of ascorbic acid in gram quantities were found to be effective in treating some cancers.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Catalase production influences germination,stress tolerance and virulence of Lecanicillium muscarium conidia

Catalases are the most important enzymatic systems used to degrade hydrogen peroxide (H₂O₂) into water and oxygen, thereby lowering intracellular hydrogen peroxide levels. Entomopathogenic fungi display increased catalase activity during germination and growth, which is necessary to counteract the hyperoxidant state produced by oxidative metabolism. We studied the influence of five different hydrocarbons on catalase production by Lecanicillium muscarium to determine the importance of catalase induction in fungal germination, stress tolerance and virulence. Conidia produced by colonies grown on different hydrocarbons showed higher rates of catalase activity compared to the control and the catalase activity of conidia produced on n-octacosane was three times higher than the activity of the control. This increase in catalase activity was accompanied by a higher level of resistance to exogenous hydrogen peroxide and a reduction in the germination time. Our study has helped to identify that increased catalase activity improves the germination and tolerance to different antioxidant stress response of L. muscarium.     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.     

Effect of Verticillium lecanii on biological characteristics and life table of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies under laboratory conditions

Effects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum , a predator of whiteflies against five different conidial concentrations (1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, and 1×10⁸ conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×10⁸ spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control.     

Human Parechovirus Genotypes -10, -13 and -15 in Pakistani Children with Acute Dehydrating Gastroenteritis

Human parechoviruses are known to cause asymptomatic to severe clinical illness predominantly respiratory and gastroenetric infections. Despite their global prevalence, epidemiological studies have not been performed in Pakistan. In this study, we retrospectively analyzed 110 fecal specimen and found 26 (24%) positive for viral RNA with HPeV-10 (n = 3, 23%), HPeV-13 (n = 4, 31%) and HPeV-15 (n = 6, 46%) genotypes. Clinical features of patients with different HPeV genotypes were compared. All HPeV positive children were aged ≤4 years (mean 13.92 months). The male-to-female ratio was 1: 1.17 (46.2 vs 53.8%) with significant association (p = .031) to HPeV infectivity. HPeV-10 and -13 were found during summer while HPeV-15 was only detected during late winter season. Disease symptoms were more severe in children infected with HPeV-10 and -13 as compared to HPeV-15. Fever and vomiting were observed in 100% cases of HPeV-10 and -13 while only 17% patients of HPeV-15 had these complaints. Phylogenetic analyses showed that HPeV-10, -13 and -15 strains found in this study have 9–13%, 16.8% and 21.8% nucleotide divergence respectively from the prototype strains and were clustered to distinct genetic lineages. This is the first report of HPeV-15 infection in humans although first identified in rhesus macaques. The arginine-glycine-aspartic acid (RGD) motif present at the C-terminal of VP1 responsible for the viral attachment to cellular integrins was not found in all of these strains. In conclusion, these findings enhance our knowledge related to the epidemiology and genetic diversity of the HPeV in Pakistan and support the need for continued laboratory based surveillance programs especially in infants and neonatal clinical settings. Further, the parechovirus pathogenesis, cross-species transmission and disease reservoirs must be ascertained to adopt better prevention measures.