A simple HPLC method for plasma level monitoring of mitotane and its two main metabolites in adrenocortical cancer patients

Mitotane (o,p'-DDD or (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane, DDD) is the drug of choice for non-resectable and metastatic adrenocortical carcinomas (ACC). Measurement of mitotane and metabolites, o,p'-DDE (1,1-dichloro-2-[p-chlorophenyl]-2-[o-chlorophenyl]ethene, DDE) and o,p'-DDA (1,1-[o,p'-dichlorodiphenyl] acetic acid, DDA) provides a better understanding of mitotane pharmacokinetics and pharmacodynamics. We have developed a simple, robust and efficient high performance liquid chromatography (HPLC) method to measure mitotane and its two main metabolites, DDE and DDA. The method involves a single ethanol extraction of mitotane, DDE, DDA, and an internal standard (int std) p,p'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) with an extraction efficiency of 77-88%. All compounds are measured simultaneously using a reversed-phase phenyl HPLC column with an isocratic elution of mobile phase at a flow rate of 0.6 ml/min followed by UV detection at λ 226 nm. Inter and intra-day validation demonstrates good reproducibility and accuracy. Limits of quantitation are 0.2 μg/ml for mitotane and DDE, and 0.5 μg/ml for DDA. The method has been evaluated in plasma from 23 patients on mitotane therapy, revealing DDA concentrations 1-18 times higher than the parent compound.     

Evidence of the formation of direct covalent adducts of primaquine, 2-tert-butylprimaquine (NP-96) and monohydroxy metabolite of NP-96 with glutathione and N-acetylcysteine

2-tert-Butylprimaquine (NP-96) is a novel quinoline anti-malarial compound with superior therapeutic profile than primaquine (PQ). Moreover, it is the first 8-aminoquinoline that is established to be devoid of methemoglobin toxicity. The purpose of the present study was to investigate covalent adduct formation tendency of PQ, NP-96 and their phase I metabolites with glutathione (GSH) and N-acetylcysteine (NAc). For the same, the two compounds were incubated in human and rat liver microsomes in the presence of trapping agents and NADPH. In a control set, NADPH was excluded, while a blank was also studied that was devoid of both NADPH and microsomes. The components in the reaction mixtures were initially separated on a C-18 column (250 mm×4.6mm, 5 μm) using a mobile phase composed of acetonitrile and 10 mM ammonium acetate in a gradient mode. The samples were then subjected to LC-MS(n) and LC-HR-MS analyses, and data were collected in full scan MS, data dependent MS/MS, targeted MS/MS, neutral loss scan (NLS) and accurate mass (MS/TOF) modes. In a significant finding, both PQ and NP-96 themselves showed potential to bind covalently with GSH and NAc, as adducts were observed even in the control and blank incubations. Intense peaks corresponding to covalent adduct of mono-hydroxy metabolite of NP-96 with GSH and NAc were also detected in NADPH supplemented reaction solution.     

Identification of haptoglobin and apolipoprotein A-I as biomarkers for high altitude pulmonary edema

We have investigated the plasma proteome using 2D gel electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight from patients with high altitude pulmonary edema (HAPE). A complete proteomic analysis was performed on 20 patients with HAPE and ten healthy sea level controls. In total, we have identified 25 protein spots in human plasma and found that 14 of them showed altered changes in HAPE patients, which mainly were acute phase proteins (APPs), compliment components, and apolipoproteins among others. Among the APPs, haptoglobin α2 chain, haptoglobin β chain, transthyretin, and plasma retinol binding precursor showed overexpression in HAPE patients as compared to controls. To validate the result of proteomic analysis, two proteins were selected for enzyme-linked immunosorbent assay and Western blotting analysis. Our data conclusively shows that two proteins, haptoglobin and apolipoprotein A-I are upregulated in plasma of HAPE patients. These proteins may provide a fast and effective control of inflammatory damage until the subsequent mechanisms can begin to operate. Taken together, our findings further support the hypothesis that inflammatory response system is linked to the pathophysiology of HAPE.     

Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Identification and validation of QTLs conferring resistance to sorghum downy mildew (Peronosclerospora sorghi) and Rajasthan downy mildew (P. heteropogoni) in maize

We have mapped the quantitative trait loci (QTLs) conferring resistance to sorghum downy mildew (Peronosclerospora sorghi; SDM) and Rajasthan downy mildew (P. heteropogoni; RDM), two species of DM prevalent throughout India. QTL mapping was carried out on a backcross population of 151 individuals derived from a cross between CM139 (susceptible parent) and NAI116 (highly resistant to both SDM and RDM). Heritability estimates were 0.74 for SDM and 0.67 for RDM. Composite interval mapping combined with a linkage map constructed with 80 simple sequence repeat (SSR) markers resulted in the identification of three QTLs (one each on chromosomes 2, 3 and 6) for SDM resistance and two QTLs (one each on chromosomes 3 and 6) for RDM resistance, all of which were contributed by NAI116. The significance of the major QTL on chromosome 6 (bin 6.05) that confers resistance to diverse DMs in tropical Asia, including SDM and RDM in India, was also verified. The results confirmed that some common QTLs contribute to both SDM and RDM resistance, while additional loci might specifically govern resistance to SDM. The QTL information generated in this study provide information that will aid in undertaking an integrated breeding strategy for the transfer of resistance to SDM and RDM in maize lines using marker-assisted selection.     

Vermistabilization of textile mill sludge spiked with poultry droppings by an epigeic earthworm Eisenia foetida

Investigations were made to explore the potential of an epigeic earthworm Eisenia foetida to transform textile mill sludge spiked with poultry droppings in to value added product, i.e., vermicompost. The growth and reproduction of E. foetida was monitored in a range of different feed mixtures for 77 days in the laboratory under controlled experimental conditions. The maximum growth was recorded in 100% cow dung (CD). Replacement of poultry droppings by cow dung in feed mixtures and vice versa had little or no effect on worm growth rate and reproduction potential. Worms grew and reproduced favourably in 70% poultry droppings (PD)+30% solid textile mill sludge (STMS) and 60% PD+40% STMS feed mixtures. Greater percentage of STMS in the feed mixture significantly affected the biomass gain and cocoon production. Net weight gain by earthworms in 100% CD was 2.9-18.2 fold higher than different STMS containing feed mixtures. The mean number of cocoon production was between 23.4+/-4.65 (in 100% CD) and 3.6+/-1.04 (in 50% PD+50% STMS) cocoons earthworm(-1) for different feed mixtures tested. Vermicomposting resulted in significant reduction in C:N ratio and increase in nitrogen and phosphorus contents. Total potassium, total calcium and heavy metals (Fe, Zn, Pb and Cd) contents were lower in the final product than initial feed mixtures. Our trials demonstrated vermicomposting as an alternate technology for the recycling and environmentally safe disposal/management of textile mill sludge using an epigeic earthworm E. foetida if mixed with poultry droppings.     

Dynamics of amino acids in the conditioning film developed on glass panels immersed in the surface seawaters of Dona Paula Bay

The conditioning film developed on glass panels immersed in surface seawater over a period of 24 h was analysed for total organic carbon (OC), total organic nitrogen (ON), and total hydrolyzable amino acid (THAA) concentrations and composition. The concentrations of C and N and THAA increased, whereas the C/N ratio decreased over the period of immersion. The amino acid-C and N accounted for 3.7-6.7% and 10.3-65.3% of OC and ON, respectively. The relative contribution of glycine plus threonine and serine to the total amino acids decreased while that of valine, phenylalanine, isoleucine and leucine increased over the period of immersion. Principal component analysis (PCA) based on mole% amino acid composition showed that the degradation indices (DI) for the conditioning film organic matter increased over the period of immersion. A high C/N ratio, a low %THAA-C, % THAA-N and DI values and the abundance of glycine plus threonine and serine in the conditioning film organic matter during the first few hours following immersion imply that the adsorbed organic matter was mostly derived from degraded organic matter.