Risk assessment of heavy metal and metalloid toxicity through a contaminated vegetable (Cucurbita maxima) from wastewater irrigated area: A case study for a site-specific risk assessment in Jhang,Pakistan

The present research was conducted in district Jhang, Pakistan, to evaluate the concentration of metals/metalloids in soil and pumpkin (Cucurbita maxima) irrigated with domestic wastewater. Data revealed that the levels of metals and metalloids in soil samples from two different sites were below the safe limits except Cd, whereas, in the vegetable, the concentrations of As, Se, Ni, Mo, Pb, Mn, and Cu were above the safe limits. The levels of 12 metals and metalloids in the soil were ranged between 0.14 to 22.76 mg/kg at site-I and 0.16 to 22.13 mg/kg at site-II. The levels of these metals in the vegetable were found 0.35 to 61.13 mg/kg at site-I and 0.31 to 53.63 mg/kg at site-II. The transfer factor at both sites was highest for As and Co. The pollution load index recorded for Se, Cu, Cd, Mo, Pb, and Co was greater than 1. The daily intake of As, Mn, and Mo was above the oral reference dose, which reflects that the intake of pumpkin is not safe for the inhabitants of the selected sites. The control measures should be taken to phytoextract heavy metals and metalloids from polluted sites so as to reduce the health risks.     

An oxygenase enzyme system for Trametes versicolor

Abstract An oxygenase enzyme was isolated from the basidiomycete fungus Trametes versicolor , that is capable of attacking lignin and a large number of di- and tri-substituted benzene rings containing at least one hydroxy group. This enzyme system was produced late in the growth cycle without the requirement for any inducer. This non-selective enzyme system is thermophilic and operates at pH 3–5 in the presence of air or oxygen. The action of this enzyme system caused the loss of UV absorption in ferulic acid solution, the formation of hydroxy muconic semialdehyde from catechol, and transformation with the production of CO₂ from a number of hydroxy aromatics as well as lignin.     

(Difluoromethylene)phosphates of guanine nucleosides as probes of DNA polymerases and G proteins

5'-Polyphosphates of N2-(p-n-butylphenyl)-2'-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5'-[beta,gamma-(Difluoromethylene)triphosphates], including that of 2'-deoxyguanosine, were prepared by reaction of the corresponding 5'-phosphates, activated by 1,1'-carbonyldiimidazole, with difluoromethanediphosphonate. The 5'-[(difluoromethylene)diphosphate] of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5'-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with 1,1'-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5'-[(alpha,beta-difluoromethylene)triphosphate]. Products were characterized by 31P and 19F NMR spectroscopy. The phosphonates were tested for their ability to displace [3H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase alpha from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF2 group was in the beta,gamma position, but not when it was in the alpha,beta position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF2 group had no effect on inhibition of DNA polymerase alpha by N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphospate]. 2'-Deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.     

Assessment of biological activities of chitosan Schiff base tagged with medicinal plants

A chitosan Schiff base with an aromatic aldehyde was synthesized and characterized by FTIR and NMR spectroscopies. Furthermore, the degree of substitution was calculated based on the ratios of the area of the proton of the imine (A_imine) and the area of the peak of the proton of the pyranose ring (A_H-2). The antimicrobial activities were determined against bacterial and fungal strains, as well as multiple drug-resistant (MDR) bacteria. The chitosan Schiff base was also tagged with medicinal plants, for example, Curcuma longa, Peganum harmala, Lepidium sativam, and cruciferous vegetables, and the biological activities determined against pathogenic bacterial and fungal strains. The chitosan Schiff base showed maximum zone of inhibition of 22 mm against Staphylococcus aureus with a minimum zone of inhibition of 15 mm against Bacillus cereus. The chitosan Schiff base was fused with C longa, isothiocyanates and a combined mixture of P harmala and L sativam that has shown activities against Escherichia coli with a zone of inhibition of 28, 24, and 30 mm, respectively. The Schiff base of chitosan fused with medicinal plants also showed significant inhibitory activities against MDR bacteria.     

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans

We previously reported the antifungal properties of a monoterpene phenol “Eugenol” against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.     

Pro-oxidant, anti-oxidant and cleavage activities on DNA of curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin.

Curcumin, a naturally occurring phytochemical responsible for the colour of turmeric shows a wide range of pharmacological properties including antioxidant, anti-inflammatory and anti-cancer effects. We have earlier shown that curcumin in the presence of Cu(II) causes strand cleavage in DNA through generation of reactive oxygen species, particularly the hydroxyl radical. Thus, curcumin shows both antioxidant as well as pro-oxidant effects. In order to understand the chemical basis of various biological properties of curcumin, we have studied the structure-activity relationship between curcumin and its two naturally occurring derivatives namely demethoxycurcumin (dmC) and bisdemethoxycurcumin (bdmC). Curcumin was found to be the most effective in the DNA cleavage reaction and a reducer of Cu(II) followed by dmC and bdmC. The rate of formation of hydroxyl radicals by the three curcuminoids also showed a similar pattern. The relative antioxidant activity was examined by studying the effect of these curcuminoids on cleavage of plasmid DNA by Fe(II)-EDTA system (hydroxyl radicals) and the generation of singlet oxygen by riboflavin. The results indicate that curcumin is considerably more active both as an antioxidant as well as an oxidative DNA cleaving agent. The DNA cleavage activity is the consequence of binding of Cu(II) to various sites on the curcumin molecule. Based on the present results, we propose three binding sites for Cu(II). Two of the sites are provided by the phenolic and methoxy groups on the two benzene rings and the third site is due to the presence of 1,3-diketone system between the rings. Furthermore, both the antioxidant as well as pro-oxidant effects of curcuminoids are determined by the same structural moieties.     

Bioproduction of benzaldehyde in a solid–liquid two-phase partitioning bioreactor using <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid–liquid two-phase partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single phase biotransformation. The two-phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid–liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.     

Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.