Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na⁺ independent; 4) saturable as a function of substrate concentration (apparent K_m = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation     

Thermal adaptability and disease association in common carp (Cyprinus carpio communis) acclimated to different (four) temperatures

The present study was designed to investigate the effect of temperature (20 °C, 24 °C, 28 °C and 32 °C) on the heamato-biochemical and histological alterations of Cyprinus carpio communis. Increase in the temperature showed significant decrease in the serum protein, while a reduced level of blood glucose at high temperature of 32 °C was observed leading to hypoglycemic conditions in the experimental fishes. A significant correlation (P<0.01) was observed between cholesterol (Cho) and triglycerides (TG) for different temperature treatments. Elevated blood urea nitrogen (BUN) at high temperatures was a good indicator of gill osmoregulatory failure. A variation of 86.40% and 38.33%, respectively, was noticed in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 32 °C over minimum experimental temperature of 20 °C. The increase in red blood cell (RBC) and Heamoglobin (Hb) concentration is associated with the decrease of mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), could be the reason for observed poikilo-anisocytosis. Histological studies of different organs of experimental fishes showed accumulation of MMC's (melanomacrophagic centers) and atrophy of the interrenal tissue on exposure to various levels of temperature. These changes were related to severity of thermal stress, being most marked when high temperature was prolonged during acclimatization. Some fishes were found infested by protozoan parasite at elevated temperature of 32 °C. Increased levels of certain biochemical and haemotological parameters studied were strongly correlated with disease in the Cyprinus carpio communis species.     

Persistent organic pollutants in Pakistan: Potential threat to ecological integrities in terms of genotoxicity and oxidative stress

As a consequence of both increasing population and industrialization in agro-economic sector, Pakistan has inevitably been confronted by multicomplex environmental challenges. Owing in part to poor regulatory framework, pollution due to persistent organic pollutants (POPs) has caused serious problems throughout the country. Resultantly, extensive use of POPs is causing vigorous deterioration of environment and human health. The current study addresses: (1) the general information on associated ecological effects and toxicity assessment by meta-analysis for local fauna and flora (2) their respective occurrence in living organisms; and (3) sources and distribution patterns of various POPs classes in environmental compartments of Pakistan. Based on the study, it can be concluded that the environment of Pakistan is highly contaminated with organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), dechlorane plus (DP), and polychlorinated naphthalenes (PCNs), which is further supported with the meta-analysis. Nevertheless, unavailability of environmental quality standards and food safety for POPs render it a forthcoming challenge of multicompartment toxicity exposure. Therefore, strategies must be planned for risk assessment of biologically active POPs, while the POP waste inventory should be elevated, along with the necessary measures to promote appropriate handling and treatment of POP as a matter of prime importance.     

Switching among natal and auxiliary hosts increases vulnerability of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) to insecticides

The role of insecticidal application and host plant resistance in managing Spodoptera exigua has been well documented, but the effect of different host plants, on which the pest cycles its population in the field, has seldom been investigated. Therefore, we have studied the vulnerability of S. exigua against commonly used insecticides (cypermethrin, chlorpyrifos, lufenuron, and emamectin benzoate) with different mode of actions when it switches its generations from natal to auxiliary hosts and vice versa. Different field populations being established on different host plants including castor, cauliflower, cotton, okra, and spinach were collected and reared in the laboratory before insecticidal bioassays. The role of larval diet and host plant switching on their response to tolerate applied insecticides was studied using leaf‐dip bioassay methods. Host switching demonstrated a significant role in altering the vulnerability of S. exigua populations to tested insecticides. Spodoptera exigua sourced from castor, when switched host to okra and spinach, exhibited 50% higher mortality when treated with emamectin benzoate. This trend in mortality was consistent upon complete host switch cycle (natal—auxiliary—natal host). However, the highest increase (92%) in vulnerability was recorded when the larvae were shifted to spinach from cotton. In general, chlorpyrifos and lufenuron had highest efficacies in terms of larval mortality. The findings of present studies provide insights to a better understanding the behavior of polyphagous pests and the role of different host plants in altering the susceptibility of these pests against applied insecticides. Ultimately the results warrant that due consideration should be given to cropping patterns and time of host switching by pest population during planning and executing chemical control.     

Taurine kinetics assessed using [1,2-13C2]taurine in healthy adult humans

To assess the dynamics of taurine metabolism in vivo, two sets of studies were carried out in healthy volunteers. First, pilot studies were carried in a single human subject to determine the time course of plasma and whole blood isotope enrichment over the course of an 8-h, unprimed continuous infusion of [1,2-(13)C(2)]taurine. Second, five healthy adult males received two tracer infusions on separate days and in randomized order: 1) a 6-h continuous infusion of [1,2-(13)C(2)]taurine (3.1 +/- 0.2 micromol x kg(-1) x h(-1)) and 2) a bolus injection of [(13)C(2)]taurine (3.0 +/- 0.1 micromol/kg). Isotope enrichments in plasma and whole blood taurine were determined by gas chromatography-mass spectrometry. The pilot experiments allowed us to establish that steady-state isotope enrichment was reached in plasma and whole blood by the 5th h of tracer infusion. The plateau enrichment reached in whole blood was lower than that obtained in plasma taurine (P < 0.02). In the second set of studies, the appearance rate (R(a)) of plasma taurine, determined from continuous infusion studies was 31.8 +/- 3.1 micromol x kg(-1) x h(-1). After a bolus injection of tracer, the enrichment decay over the subsequent 2 h was best fitted by a two-exponential curve. Taurine R(a) was approximately 85% higher when determined using the bolus injection technique compared with continuous infusion of tracer. We conclude that 1) taurine R(a) into plasma is very low in healthy postabsorptive humans, and, due to taurine compartmentation between the extra- and intracellular milieus, may represent only interorgan taurine transfer and merely a small fraction of whole body taurine turnover; and 2) the bolus injection technique may overestimate taurine appearance into plasma. Further studies are warranted to determine whether alterations in bile taurine dynamics affect taurine R(a).     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

An 18 kDa Acid Phosphatase from Chicken Heart Possesses Phosphotransferase Activity

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K _cat/K _m, 4.3×10⁴, found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.     

Correlation between decreased sensitivity of the Daudi lymphoma cells to VP-16-induced apoptosis and deficiency in DNAS1L3 expression

A functional relationship between the apoptotic endonuclease DNAS1L3 and the chemotherapeutic drug VP-16 was established. The lymphoma cell line, Daudi, exhibited a significant resistance to VP-16 treatment in comparison to the lymphoma/leukemia cell line, U-937. While U-937 cells degraded their DNA into internucleosomal fragments, Daudi cells failed to undergo such fragmentation in response to the drug. Activation of both caspase-3 and DNA fragmentation factor was not sufficient to trigger internucleosomal DNA fragmentation in Daudi cells. No correlation was found between expression levels of topoisomerase-II, Pgp, Bcl-2, Bax, or Bad and decreased sensitivity of Daudi cells to VP-16. Daudi cells failed to express DNAS1L3 and ectopic expression of this protein significantly sensitized the cells to VP-16. An enhancement of caspase-3 activity and collapse of mitochondrial membrane potential underlie DNAS1L3-mediated sensitization of Daudi cells to VP-16, which may be a direct result of DNAS1L3-mediated increase in PARP-1-activating DNA breaks after VP-16 treatment. Our results suggest that DNAS1L3 plays an active role in lymphoma cell sensitization to VP-16 and that its deficiency may constitute a novel mechanism of drug resistance in these cells.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.