To determine the survival, prevalence and associated factors of exclusive breastfeeding practices in South India.

Context: Exclusive breastfeeding (EBF) provides protection to the child from risk of obesity, overweight, type II diabetes and helps in enhancing brain development, learning capabilities and also reduces gastrointestinal infections. Breast problems, societal barriers, insufficient support, poor knowledge, mode of delivery and community beliefs are associated. Aim: To determine the survival and prevalence of exclusive breast-feeding practice and their associated factors. Settings and Design: An ambispective community-based observational study was conducted. Materials and Methods: A sample of 441 mothers was estimated with a prevalence of EBF of 54.9% based on the National Family Health Survey 2015-16. Study Procedure: The selected mothers with an infant less than one year of age and those with infants less than six months were interviewed retrospectively and prospectively and information on the duration of EBF, demography and factors associated were collected. Statistical Analysis Used: The data were analysed using IBM SPSS, version 22. The Chi-square test and binary logistic regression were used to determine the associated factors for EBF. A P value of <0.05 was considered significant. Results: EBF survival rate was good till three months and decreased drastically after five months. EBF practice in the present study was 69.4%. Birth order, maternal age, birth weight, paternal education and religion were significantly associated with EBF. Conclusion: Primary health care providers in the community should also consider the cultural factors and educate the mothers on the practice of EBF to reduce morbidity and mortality and promote better health for a healthy, strong, younger population.

Maternal and Child Health Pipeline Training Programs: A Description of Training Across 6 Funded Programs.

PURPOSE: The HRSA-funded maternal and child health pipeline training programs (MCHPTPs) are a response to the critical need to diversify the MCH workforce, as a strategy to reduce health disparities in MCH populations. These MCHPTPs support students from undergraduate to graduate education and ultimately into the MCH workforce. DESCRIPTION: The models and components of training across the six MCHPTPs funded in 2016-2021 are summarized, to examine the design and delivery of undergraduate pipeline training and the insights gained across programs. ASSESSMENT: Strategies that emerged across training programs were organized into three themes: recruitment, support for student persistence (in education), and pipeline-to-workforce intentionality. Support for student persistence included financial support, mentoring, creating opportunity for students to develop a sense of belonging, and the use of research as a tool to promote learning and competitiveness for graduate education. Finally, the link to Maternal and Child Health Bureau (MCHB) long-term training and other MCHB opportunities for professional development contributed significant nuance to the pipeline-to-workforce objectives of these programs. CONCLUSIONS: The MCHPTPs not only increase the diversity of the MCH workforce, they also actively prepare the next generation of MCH leaders. The intentional connection of undergraduates to the infrastructure and continuum of MCH training, underscores the comprehensive impact of this funding.

Unconditional probability of dying and age-specific mortality rate because of major non-communicable diseases in India: Time trends from 2001 to 2013.

Background: Unconditional probability of dying because of four major non-communicable diseases (NCDs) between 30 and 70 years of age is the selected global indicator to measure the impact of NCD prevention and control programs. Objective: To calculate the unconditional probability of dying and age-specific mortality rate because of major NCDs in India from 2001 to 2013. Methods: This study used multiple data sources that are available in the public domain-Census 2001 and 2011, Sample Registration System, causes of death reports in 2001-03, 2004-06, and 2010-13. Unconditional probability of dying between ages 30 and 70 years during 2001, 2006, and 2013 was calculated by the formula suggested by the World Health Organization. Line graphs were used to depict time trends in age-specific mortality rates over the years in four major NCDs (cardiovascular diseases, cancer, diabetes, and chronic respiratory diseases). Results: The age-specific mortality rate because of four NCDs showed a decrease of 51 deaths per 100,000 population from 2001 to 2013. Of the four NCDs, age-specific mortality rate was highest in cardiovascular diseases (238.2/100,000 population) and least in diabetes mellitus (21.9/100,000 population); it was 76.3 and 58.2/100,000 population for cancer and chronic respiratory diseases, respectively. The probability of dying was very less and was almost the same from 30 to 44 years of life and increased steeply after that till 70 years of life; and it was more in males (24%) compared with females (17.4%). Conclusion: Although India has shown a decreasing trend in premature mortality because of NCDs in the past decade, the rate of decrease is not on par to achieve the global "25 × 25" target.

Status of fluid and electrolyte absorption in cystic fibrosis.

Salt and fluid absorption is a shared function of many of the body's epithelia, but its use is highly adapted to the varied physiological roles of epithelia-lined organs. These functions vary from control of hydration of outward-facing epithelial surfaces to conservation and regulation of total body volume. In the most general context, salt and fluid absorption is driven by active Na(+) absorption. Cl(-) is absorbed passively through various available paths in response to the electrical driving force that results from active Na(+) absorption. Absorption of salt creates a concentration gradient that causes water to be absorbed passively, provided the epithelium is water permeable. Key differences notwithstanding, the transport elements used for salt and fluid absorption are broadly similar in diverse epithelia, but the regulation of these elements enables salt absorption to be tailored to very different physiological needs. Here we focus on salt absorption by exocrine glands and airway epithelia. In cystic fibrosis, salt and fluid absorption by gland duct epithelia is effectively prevented by the loss of cystic fibrosis transmembrane conductance regulator (CFTR). In airway epithelia, salt and fluid absorption persists, in the absence of CFTR-mediated Cl(-) secretion. The contrast of these tissue-specific changes in CF tissues is illustrative of how salt and fluid absorption is differentially regulated to accomplish tissue-specific physiological objectives.

Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms.

The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.

The JAK2V617F oncogene requires expression of inducible phosphofructokinase/fructose-bisphosphatase 3 for cell growth and increased metabolic activity.

Myeloproliferative neoplasms are characterized by overproduction of myeloid lineage cells with frequent acquisition of oncogenic JAK2V617F kinase mutations. The molecular mechanisms that regulate energy requirements in these diseases are poorly understood. Transformed cells tend to rely on fermentation instead of more efficient oxidative phosphorylation for energy production. Our data in JAK2V617F-transformed cells show that growth and metabolic activity were strictly dependent on the presence of glucose. Uptake of glucose and cell surface expression of the glucose transporter Glut1 required the oncogenic tyrosine kinase. Importantly, JAK2V617F as well as active STAT5 increased the expression of the inducible rate-limiting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which controls glycolytic flux through 6-phosphofructo-1-kinase. PFKFB3 was required for JAK2V617F-dependent lactate production, oxidative metabolic activity and glucose uptake. Targeted knockdown of PFKFB3 also limited cell growth under normoxic and hypoxic conditions and blocked in vivo tumor formation in mice. Overall, these data suggest that inducible PFKFB3 is required for increased growth, metabolic activity and is regulated through active JAK2 and STAT5. Novel therapies that specifically block PFKFB3 activity or expression would, therefore, be expected to inhibit JAK2/STAT5-dependent malignancies and related cancers.

NADPH oxidases regulate cell growth and migration in myeloid cells transformed by oncogenic tyrosine kinases.

Transformation by tyrosine kinase oncogenes (TKOs) in myeloid malignancies, including BCR-ABL in chronic myeloid leukemia, FLT3ITD in acute myeloid leukemia or JAK2V617F in myeloproliferative neoplasms, is associated with increased growth and cytoskeletal abnormalities. Using targeted approaches against components of the superoxide-producing NADPH-oxidases, including NADPH oxidase 2 (NOX2), NOX4 and the common p22(phox) subunit of NOX1-4, myeloid cells were found to display reduced cell growth and spontaneous migration. Consistent with a role of NOXs as regulators of membrane proximal signaling events in nonphagocytic cells, NOX2 and NOX4 were not involved in the excess production of intracellular reactive oxygen species and did not significantly increase oxygen consumption. All NOX family members are controlled in part through levels of the rate-limiting substrate NADPH, which was found to be significantly elevated in TKO-transformed cells. Also, reduced phosphorylation of the actin filament crosslinking protein myristoylated alanine-rich C-kinase substrate (MARCKS) in response to suppression of p22(phox) hints at a novel effector of NOX signaling. MARCKS was also found to be required for increased migration. Overall, these data suggest a model whereby NOX links metabolic NADPH production to cellular events that directly contribute to transformation.

PKA mediates constitutive activation of CFTR in human sweat duct.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.

Induction of hsp70, hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed Drosophila melanogaster: role of ROS generation.

Exposure to benzene, toluene and xylene in the human population may pose a health risk. We tested a working hypothesis that these test chemicals cause cellular toxicity to a non-target organism, Drosophila melanogaster. Third instar larvae of D. melanogaster transgenic for hsp70, hsp83 and hsp26 and Oregon R(+) strain were exposed to 1.0-100.0 mM benzene, toluene and xylene for 2-48 h to examine the heat shock proteins (hsps), ROS generation, anti-oxidant stress markers and developmental end points. The test chemicals elicited a concentration- and time-dependent significant (p<0.01) induction of the hsps in the exposed organism in the order of hsp70>hsp83>or=hsp26 as evident by beta-galactosidase activity after 24 h. RT-PCR amplification studies in Oregon R(+) larvae revealed a similar induction pattern of these genes along with hsp60 in the order of hsp70>hsp60>hsp26>or=hsp83. Under similar experimental conditions, a significant induction of ROS generation and oxidative stress markers viz. superoxide dismutase, catalase, glutathione S-transferase, thioredoxin reductase, glutathione, malondialdehyde and protein carbonyl content was observed. Sub-organismal response was propagated towards organismal response i.e., a delay in the emergence of flies and their reproductive performance. While hsp70 was predominantly induced in the organism till 24 h of treatment with the test chemicals, a significant or insignificant regression of Hsp70 after 48 h was concurrent with a significant induction (p<0.01) of hsp60>hsp83>or=hsp26 in comparison to the former. A significant positive correlation was observed between ROS generation and these hsps in the exposed organism till 24 h and a negative correlation between ROS generation and hsp70 in them after 48 h indicating a modulatory role of ROS in the induction of hsps. The study suggests that among the tested hsps, hsp70 may be used as an early bioindicator of cellular toxicity against benzene, toluene and xylene and D. melanogaster as an alternative animal model for screening the risk posed by environmental chemicals.

Effect of cytosolic pH on epithelial Na+ channel in normal and cystic fibrosis sweat ducts.

The activities of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and the amiloride-sensitive epithelial Na(+) channel (ENaC) are acutely coordinated in the sweat duct. However, the mechanisms responsible for cross-talk between these ion channels are unknown. Previous studies indicated that luminal pH of sweat ducts varies over 3 pH units and that the cytoplasmic pH affects both CFTR and ENaC. Therefore, using basolaterally alpha-toxin-permeabilized apical membrane preparations of sweat ducts as an experimental system, we tested the hypothesis that the cytosolic pH may mediate the cross-talk between CFTR and ENaC. We showed that while luminal pH had no effect, cytosolic pH acutely affected ENaC activity. That is, acidic pH inhibited, while basic pH activated, ENaC. pH regulation of ENaC appears to be independent of CFTR or endogenous kinase activities because basic pH independently stimulated ENaC (1) in normal ducts even when CFTR was deactivated, (2) in CF ducts that lack CFTR in the plasma membranes and (3) after blocking endogenous kinase activity with staurosporine. Considering the evidence of Na(+)/H(+) exchange (NHE) activity as shown by the expression of mRNA and function of NHE in the basolateral membrane of the sweat duct, we postulate that changes in cytosolic Na(+) ([Na(+)]( i )) may alter cytosolic pH (pH( i )) as salt loads into the cell during electrolyte absorption. These changes may play a role in coordinating the activities of ENaC and CFTR during transepithelial salt transport.

Iontophoretic beta-adrenergic stimulation of human sweat glands: possible assay for cystic fibrosis transmembrane conductance regulator activity in vivo.

With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished beta-adrenergic but not cholinergic sweating in CF. Therefore, the beta-adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating beta-adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in beta-adrenergic and cholinergic sweating. We also tested the efficacy of different beta-adrenergic agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the beta-adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by beta-adrenergic agonists are significantly smaller compared with those generated by cholinergic agonists, which in turn reflects in smaller beta-adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the beta-adrenergic agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent beta-adrenergic sweat responses, levels of intracellular cAMP above that achievable with a beta-adrenergic agonist alone are essential. beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.

Isolation and characterization of bacterial strains Paenibacillus sp. and Bacillus sp. for kraft lignin decolorization from pulp paper mill waste.

Eight aerobic bacterial strains were isolated from pulp paper mill waste and screened for tolerance of kraft lignin (KL) using the nutrient enrichment technique in mineral salt media (MSM) agar plate (15 g/L) amended with different concentrations of KL (100, 200, 300, 400, 500, 600 ppm) along with 1% glucose and 0.5% peptone (w/v) as additional carbon and nitrogen sources. The strains ITRC S6 and ITRC S8 were found to have the most potential for tolerance of the highest concentration of KL. These organisms were characterized by biochemical tests and further 16S rRNA gene (rDNA) sequencing, which showed 96.5% and 95% sequence similarity of ITRC S(6) and ITRC S(8) and confirmed them as Paenibacillus sp. and Bacillus sp., respectively. KL decolorization was routinely monitored with a spectrophotometer and further confirmed by HPLC analysis. Among eight strains, ITRC S(6) and ITRC S(8) were found to degrade 500 mg/L of KL up to 47.97% and 65.58%, respectively, within 144 h of incubation in the presence of 1% glucose and 0.5% (w/v) peptone as a supplementary source of carbon and nitrogen. In the absence of glucose and peptone, these bacteria were unable to utilize KL. The analysis of lignin degradation products by GC-MS analysis revealed the formation of various acids as lignin monomers which resulted in a decrease in pH and a major change in the chromatographic profile of the bacterial degraded sample as compared to the control clear indications of biochemical modification of KL due to the bacterial ligninolytic system by ITRC S(6), namely, acetic acid, propanoic acid, butanoic acid, guaiacol, hexanoic acid, and ITRC S(8), namely acetic acid, propanoic acid, ethanedioic acid, furan carboxylic acid, 2-propanoic acid, butanoic acid, 3-acetoxybutyric acid, propanedioic acid, acetoguiacone, 1,2,3-thiadiazole, 5-carboxaldixime, 4-hydroxy-3,5-dimethoxyphenol, and dibutyl phthalate, indicating the bacterium characteristic to degrade G and S units of lignin polymer.

Investigation of the biotransformation of pentachlorophenol and pulp paper mill effluent decolorisation by the bacterial strains in a mixed culture.

Mixed culture of two bacterial strains Bacillus sp. and Serratia marcescens showed potential pentachlorophenol (PCP) degradation and decolorisation of pulp paper mill effluent. The physico-chemical quality of pulp paper mill effluent has been analyzed after 168 h incubation period degraded by mixed culture. The study revealed that it has decreased high load of BOD, COD, TS, TDS, TSS, sulphate, phosphate, total nitrogen, total phenols, metals and different salts (i.e. chloride, sodium, nitrate, potassium) at 168 h incubation period. PCP degradation in pulp paper mill effluent was confirmed by HPLC analysis. Mixed culture was found to degrade PCP up to (94%) present in pulp paper mill effluent with 1% glucose and 0.5% peptone (w/v) at 30+/-1 degrees C, pH 8.0+/-0.2 at 120 rpm in 168 h incubation period. The simultaneous release of chloride ion up to 1,200 mg/l at 168 h emphasized the bacterial dechlorination in the medium. The pulp paper mill effluent degradation was also supported by decline in pH, AOX (absorbable organic halides), color, D.O., BOD, COD and PCP. The analysis of pulp paper mill effluent degradation products by GC-MS analysis revealed the formation of low molecular weight compound like 2-chlorophenol (RT=3.8 min) and tetrachlorohydroquinone (RT=11.86 min) from PCP extracted degraded sample. Further, mixed culture may be used for bioremediation of PCP containing pulp paper mill waste in the environment.

Biodegradation of kraft-lignin by Bacillus sp. isolated from sludge of pulp and paper mill.

Eight bacterial strains were isolated on kraft lignin (KL) containing mineral salt medium (L-MSM) agar with glucose and peptone from the sludge of pulp and paper mill. Out of these, ITRC-S8 was selected for KL degradation, because of its fast growth at highest tested KL concentration and use of various lignin-related low molecular weight aromatic compounds (LMWACs) as sole source of carbon and energy. The bacterium was identified by biochemical tests as Gram-positive, rod-shaped and non-motile. Subsequent 16S rRNA gene sequencing showed 95% base sequence homology and it was identified as Bacillus sp. In batch experiments, a decrease in pH was observed initially followed by an increase till it reached an alkaline pH, which did not alter the culture growth significantly. The bacterium reduced the colour and KL content of 500 mg l(-1 )KL in MSM, in the presence of glucose and peptone, at pH 7.6, temperature 30 degrees C, agitation of 120 rpm and 6 days of incubation by 65 and 37% respectively. Significant reduction in colour and KL content in subsequent incubations is indicative of a co-metabolism mechanism, possibly due to initial utilization of added co-substrates for energy followed by utilization of KL as a co-metabolic. The degradation of KL by bacterium was confirmed by GC-MS analysis indicating formation of several LMWACs such as t-cinnamic acid, 3, 4, 5-trimethoxy benzaldehyde and ferulic acid as degradation products, which were not present in the control (uninoculated) sample. This favours the idea of biochemical modification of the KL polymer to a single monomer unit.

Biomonitoring of organochlorines, glutathione, lipid peroxidation and cholinesterase activity among pesticide sprayers in mango orchards.

BACKGROUND: Pesticide sprayers in mango orchards of Malihabad, Lucknow (India) are generally exposed to organophosphate (OP) and pyrethroid pesticides. We determined the pesticide exposure levels along with their biochemical and clinical effects in 31 sprayers, compared with 18 controls. METHODS: Assay of acetyl and butyrylcholinesterases (AChE, BChE respectively) as an indirect measurement of OP exposure and levels of malondialdehyde (MDA) and glutathione (GSH) were estimated in blood samples to determine their impact on redox potential. Organochlorines were estimated by GLC-ECD. RESULTS: Significantly inhibited AChE, BChE activities and higher MDA level were found among sprayers compared to controls (p<0.05). Mean of total organochlorines were surprisingly higher (97.65+/-13.38 ppb) in sprayers than in those of controls (20.42+/-3.56 ppb) (p<0.05). Respiratory morbidity (32.4%), ocular problems (8.8%), gastrointestinal (17.6%) and skin problems (23.5%) were found in sprayers. There was significant correlation between AChE and GSH (r=0.29, p<0.05) and AChE with MDA (r=-0.34, p<0.05). CONCLUSION: Results indicated the significantly enhanced lipid peroxidation in sprayers correlated with cholinesterases inhibition. A small sample size limits the significance of this study. However, it paves the way for a larger Indian study with extended practical significance.

Possible mechanism of pesticide toxicity-related oxidative stress leading to airway narrowing.

The study was conducted to assess the magnitude of oxidative stress and lung function abnormalities in 34 male pesticide sprayers on exposure to pesticides in mango plantations. Biochemical studies on blood antioxidant enzymes revealed an unchanged glutathione level and increased level of malondialdehyde (P < 0.001), which indicates that pesticide sprayers may have suffered from oxidative stress. Decreased acetyl-cholinesterase levels (P < 0.001) in sprayers compared to the controls suggest inhibition of cholinesterase activity. The present study shows that pesticide toxicity might lead to oxidative stress and airway narrowing resulting in decreased peak expiratory flow rate.

Cytosolic potassium controls CFTR deactivation in human sweat duct.

Absorptive epithelial cells must admit large quantities of salt (NaCl) during the transport process. How these cells avoid swelling to protect functional integrity in the face of massive salt influx is a fundamental, unresolved problem. A special preparation of the human sweat duct provides critical insights into this crucial issue. We now show that negative feedback control of apical salt influx by regulating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity is key to this protection. As part of this control process, we report a new physiological role of K(+) in intracellular signaling and provide the first direct evidence of acute in vivo regulation of CFTR dephosphorylation activity. We show that cytosolic K(+) concentration ([K(+)](c)) declines as a function of increasing cellular NaCl content at the onset of absorptive activity. Declining [K(+)](c) cause parallel deactivation of CFTR by dephosphorylation, thereby limiting apical influx of Cl(-) (and its co-ion Na(+)) until [K(+)](c) is stabilized. We surmise that [K(+)](c) stabilizes when Na(+) influx decreases to a level equal to its efflux through the basolateral Na(+)-K(+) pump thereby preventing disruptive changes in cell volume.

Source identification of Indian opium based on chromatographic fingerprinting of amino acids.

Total and free pool of amino acids was determined in Indian opium samples using liquid chromatography (LC) with post-column opthalaldehyde derivatization followed by its fluorimetric detection. The limit of detection (LOD) was found to be in the range of 2-10 pmol with a signal to noise ratio of 3:1 and limit of quantitation (LOQ) was found to be in the range of 7-31 pmol with a signal to noise ratio of 10:1. The recovery of amino acids was found to be in the range of 86-103%. A total of 124 Indian opium samples were collected from the states of Madhya Pradesh (MP), Uttar Pradesh (UP) and Rajasthan (Raj), covering 14 licit opium growing divisions of India were chromatographically fingerprinted for the presence of various amino acids. The amino acids identified in sample hydrosylate included D, T, S, S, G, A, V, I, L, Y, F, H, K and R, while the analysis of free pool of amino acids (80% aqueous ethanol extract) indicated the presence of D, T, S, E, A, V, I, L, Y, H, K respectively. Multiple discriminant analysis was applied to the quantitative total amino acid data to determine an optimal classifier in order to evaluate the source of Indian opium. The foremost amino acid variables that accounted for the true discrimination were identified as D, E, G, A, F and K in evaluating the geographical origin of Indian opium and the predictive value based on the discriminant analysis was found to be 90% in relation to the source of opium samples. Chemometrics performed with amino acid analytical data was used successfully in discriminating the licit opium growing divisions of India into three major groups, viz. groups I, II and III. The methodology developed may find wide application in forensic analysis.

Normal CFTR Activity and Reversed Skin Potentials in Pseudohypoaldosteronism.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel function is required for activating amiloride-sensitive epithelial Na(+) channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 +/- 10 ms, mean +/- SE: ). After permeabilization of the basolateral membrane with alpha-toxin, cAMP + ATP activation of CFTR Cl(-) conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (-13 +/- 7.0 mV) to significantly positive (+22 +/- 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism.

ENaC activity requires CFTR channel function independently of phosphorylation in sweat duct.

We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl- conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na+ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl(-) -channel function is required for activation. We used basilaterally alpha-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl(-) -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl- channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 microM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl- current (I(Cl-)) through CFTR was blocked by: 1) Cl- removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 microM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous DeltaF508 CF ducts). However, Cl- gradients in the direction of absorption supported, while Cl- gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated I(Cl-) through CFTR in the direction of absorption (Cl- gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing I(Cl-) through CFTR in the direction of secretion (Cl- gradient from cell to lumen) prevents ENaC activation even in the presence of I(Cl-) through CFTR.