Rapid Filtration Technique Research Articles

Removal of Manganese and Iron from Groundwater by using Chlorination and Rapid Sand Filtration: A Case Study

The average groundwater extraction from Murunkan well field, Mannar, is around 3.4 MCM/year, and it was triggered by extensive development projects and resettlements that took place in the last decade. Manganese and Iron variations in Murunkan groundwater reported during some periods created various operational and aesthetic issues in the potable water supply. This study is focused on treating iron and manganese in groundwater using chlorination and Rapid Sand Filtration (RSF) techniques. Pebble layers, height of 205 mm with the particle sizes ranging from 2-25 mm, were filled at the bottom of RSF model filter. Filter media, height of 700 mm with 0.45 mm effective size (D10) and a uniformity coefficient (Cu) of 1.33, was placed above the pebble layers. The filter was operated continuously for 15 days, and influent and effluent water samples were collected from 15 trials. Filter backwashing was performed after every two trials to maintain a consistent filtration rate. Raw water pH values ranged from 6.74 – 7.48. Chlorine solution (1.08-1.26 ppm) was added at the inlet point, and a 2.88 m water column was maintained, allowing adequate mixing and oxidation time. The results revealed that average removals of iron and manganese as 60% and 73%, respectively, while maximum removals of 93% and 91% were observed for iron and manganese, respectively.

Decreased placental folate transporter expression and activity in first and second trimester in obese mothers

Obese women have an approximately twofold higher risk to deliver an infant with neural tube defects (NTDs) despite folate supplementation. Placental transfer of folate is mediated by folate receptor alpha (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC). Decreased placental transport may contribute to NTDs in obese women. Serum folate levels were measured and placental tissue was collected from 13 women with normal BMI (21.9±1.9) and 11 obese women (BMI 33.1±2.8) undergoing elective termination at 8–22 weeks of gestation. The syncytiotrophoblast microvillous plasma membranes (MVM) were isolated using homogenization, magnesium precipitation, and differential centrifugation. MVM expression of FR-α, PCFT and RFC was determined by western blot. Folate transport capacity was assessed using radiolabeled methyl-tetrahydrofolate and rapid filtration techniques. Differences in expression and transport capacity were adjusted for gestational age and maternal age in multivariable regression models. P<.05 was considered statistically significant. Serum folate levels were not significantly different between groups. Placental MVM folate transporter expression did not change with gestational age. MVM RFC (−19%) and FR-α (−17%) expression was significantly reduced in placentas from obese women (P<.05). MVM folate transporter activity was reduced by−52% (P<.05) in obese women. These differences remained after adjustment for gestational age. There was no difference in mTOR signaling between groups. In conclusion, RFC and FR alpha expression and transporter activity in the placental MVM are significantly reduced in obese women in early pregnancy. These results may explain the higher incidence of NTDs in infants of obese women with adequate serum folate.

A field-deployable surface-enhanced Raman scattering (SERS) method for sensitive analysis of silver nanoparticles in environmental waters

The proliferation of silver nanoparticles (AgNPs) in the environment and resultant transport into aquatic systems have raised concerns regarding their potential toxicity to various organisms. These environmental and ecological concerns demand reliable AgNP detection methods which can measure environmentally relevant quantities of AgNPs in real aquatic systems. This study developed a method that couples a rapid vacuum filtration technique with a portable Raman spectrometer to achieve on-site detection of ultra-low levels of AgNPs in typical and complex aquatic systems. To extract and detect AgNPs, aluminum chloride and ferbam were added for AgNP aggregation and labelling, respectively. The AgNP aggregates were filtered through a membrane, and their presence and quantity were determined based upon the surface-enhanced Raman scattering (SERS) peak intensity of ferbam. Under the optimized conditions, the extraction efficiencies are 99 ± 0.001% in ultrapure water and 98 ± 0.025% in marine water for 1 mg/L AgNPs. This method enables simple volume adjustment and improves the consistency of AgNP distribution on the membrane. The performance of the method was evaluated in different environmental waters, including marine water, fresh waters (pond water, river water, and reservoir outlet water) and drinking waters (municipal tap water and well water), with highest signal intensity in marine water and lowest signals in fresh waters. The signal intensity difference was suggested to be caused by the amount of natural organic matter (NOM) in these environmental waters. Using pond water as an example, the interference was minimized by changing the aggregating salt from AlCl3 to MgCl2, and AgNPs as low as 5 μg/L were reliably detected with a volume of 100 mL. At the same volume, the developed method was sensitive enough to detect 1 μg/L AgNPs in marine water and also holds promise for assessing the time-dependent transformation of AgNPs.

Reduced placental amino acid transport in response to maternal nutrient restriction in the baboon.

Intrauterine growth restriction increases the risk of perinatal complications and predisposes the infant to diabetes and cardiovascular disease in later life. Mechanisms by which maternal nutrient restriction (MNR) reduces fetal growth are poorly understood. We hypothesized that MNR decreases placental amino acid (AA) transporter activity, leading to reduced transplacental transfer of AAs. Pregnant baboons were fed either a control (ad libitum, n = 7), or MNR diet (70% of control diet, n = 7) from gestational day (GD) 30. At GD 165 (0.9 gestation), placentas (n = 7 in each group) were collected, and microvillous plasma membrane vesicles (MVM) isolated. MVM system A and system L AA transport was determined in vitro using radiolabeled substrates and rapid filtration techniques. In vivo transplacental AA transport was assessed by infusing nine (13)C- or (2)H-labeled essential AA as a bolus into the maternal circulation (n = 5 control, n = 4 MNR) at cesarean section. A fetal vein-to-maternal artery mole percent excess ratio for each essential AA was calculated. Fetal and placental weights were significantly reduced in the MNR group compared with controls (P < 0.01). The activity of system A and system L was markedly reduced by 73 and 84%, respectively, in MVM isolated from baboon placentas at GD 165 following MNR (P < 0.01). In vivo, the fetal vein-to-maternal artery mole percent excess ratio was significantly reduced for leucine, isoleucine, methionine, phenylalanine, threonine, and tryptophan in MNR baboons (P < 0.05). This is the first study to investigate placental AA transport in a nonhuman primate model of MNR. We demonstrate that the downregulation of system A and system L activity in syncytiotrophoblast MVM in MNR leads to decreased transplacental AA transport and, consequently, reduced circulating fetal AA concentrations, a potential mechanism linking maternal undernutrition to reduced fetal growth.

Mast cell regulation of Na-glutamine co-transporters B0AT1 in villus and SN2 in crypt cells during chronic intestinal inflammation.

BackgroundIn the chronically inflamed rabbit small intestine, brush border membrane (BBM) Na-glutamine co-transport is inhibited in villus cells (mediated by B0AT1), while it is stimulated in crypt cells (mediated by SN2/SNAT5). How mast cells, known to be enhanced in the chronically inflamed intestine, may regulate B0AT1 in villus and SN2/SNAT5 in crypt cell is unknown. Thus, the aim of the present study is to determine the regulation of B0AT1 and SN2/SNAT5 by mast cells during chronic enteritis.MethodsChronic intestinal inflammation was induced in male rabbits with intra-gastric inoculation of Eimeria magna oocytes. Rabbits with chronic inflammation were treated with ketotifen (10 mg/day) or saline (Placebo) for 2 days. Villus and crypts cells were isolated from the rabbit intestine using the Ca++ chelation technique. Na/K-ATPase activity was measured as Pi from cellular homogenate. BBM vesicles (BBMV) were prepared from villus and crypt cells and uptake studies were performed using rapid filtration technique with 3H-Glutamine. Western blot analyses were done using B0AT1 and SN2 specific antibodies.ResultsIn villus cells, Na-glutamine co-transport inhibition observed during inflammation was completely reversed by ketotifen, a mast cell stabilizer. In contrast, in crypt cells, Na-glutamine co-transport stimulation was reversed to normal levels by ketotifen. Kinetic studies demonstrated that ketotifen reversed the inhibition of B0AT1 in villus cells by restoring co-transporter numbers in the BBM, whereas the stimulation of SN2/SNAT5 in crypts cells was reversed secondary to restoration of affinity of the co-transporter. Western blot analysis showed that ketotifen restored immune-reactive levels of B0AT1 in villus cells, while SN2/SNAT5 levels from crypts cell remained unchanged.ConclusionIn the present study we demonstrate that mast cells likely function as a common upstream immune pathway regulator of the Na-dependent glutamine co-transporters, B0AT1 in villus cells and SN2 in crypts cells that are uniquely altered in the chronically inflamed small intestine.

Influence of aminoglycoside antibiotics on chicken cystatin binding to renal brush-border membranes

Drug-induced kidney injury is a serious adverse event which needs to be monitored during aminoglycoside therapy. Urine cystatin C is considered an early and sensitive marker of nephrotoxicity. Cystatin C, a low-molecular-weight serum protein, and basic drugs have a common transport system expressed in the apical membrane of renal proximal tubular cells. The aim of this study was to investigate whether aminoglycoside antibiotics influenced cystatin C binding to the renal brush-border membrane. The binding study was performed using a rapid filtration technique and affinity column displacement method. Concentration-dependent inhibition of chicken cystatin binding to brush-border membranes by gentamicin was observed. The gentamicin interaction with brush-border membranes was of relatively low affinity (Ki = 32 μm) in comparison with the chicken cystatin affinity to the binding sites (Kd = 3.6 μm). Amikacin and gentamicin were only able to displace chicken cystatin from the chromatographic affinity column in concentrations several times higher than normally found in the tubular fluid during standard aminoglycoside therapy. Cystatin reabsorption in the proximal tubule cannot be significantly affected by aminoglycoside antibiotics because of their relatively low affinity to common binding sites on the brush-border membrane.

Development of rapid methodologies for the isolation and quantitation of drug metabolites by differential mobility spectrometry – mass spectrometry

Clinical and forensic toxicology laboratories are inundated with thousands of samples requiring lengthy chromatographic separations prior to mass spectrometry. Here, we employ differential mobility spectrometry (DMS) interfaced to nano-electrospray ionization-mass spectrometry to provide a rapid ion filtration technique for the separation of ions in gas phase media prior to mass spectral analysis on a DMS-integrated AB SCIEX API 3000 triple-quadrupole mass spectrometer. DMS is efficient at the rapid separation of ions under ambient conditions and provides many advantages when used as an ion filtration technique in tandem with mass spectrometry (MS) and MS/MS. Our studies evaluated DMS-MS/MS as a rapid, quantitative platform for the analysis of drug metabolites isolated from urine samples. In targeted applications, five metabolites of common drugs of abuse were effectively and rapidly separated using isopropanol and ethyl acetate as transport gas modifiers, eliminating the gas chromatography or liquid chromatography-based separations commonly employed in clinical and forensic toxicology laboratories. Calibration curves were prepared for the selected drug metabolites utilizing deuterated internal standards for quantitative purposes. The feasibility of separating and quantitating drug metabolites in a rapid fashion was evaluated by compensation voltage stepping followed by multiple reaction monitoring (MRM) detection. Rapid profiling of clinical and forensic toxicology samples could help to address an urgent need within the scientific community by developing high-throughput analytical methodologies, which could reduce significant case backlogs present within these laboratories.

Characterization of L-lysine transport across equine and porcine jejunal and colonic brush border membrane

In nonruminant herbivores, microbially derived AA could contribute to whole-body AA homeostasis and thus decrease predicted AA requirements. However, postileal capacity of AA uptake is currently unknown. Therefore, to test the hypothesis that Lys is transported across the large colon mucosal apical membrane with capacity similar to that of the small intestinal mucosa in the pony and pig, we examined Lys transport in vitro using brush border membrane vesicles (BBMV). Mucosa was collected from the distal jejunum (DJ) and proximal large colon (PLC) of growing pigs (n = 3) and ponies (n = 4), flash frozen in liquid nitrogen, and stored at -80°C. Jejunal and colonic BBMV were manufactured by Mg(2+) precipitation and used to determine initial rates and kinetics [the maximal transport rate (V(max)) and the Michaelis constant (K(M))] of l-Lys transport into apical epithelia by rapid filtration technique in Na(+)-gradient incubation buffer. Initial rates of total l-Lys uptake did not differ between the PLC and DJ in either the pig or the pony, or between the pony and the pig, at each l-Lys concentration. In the pig, compared with the DJ, l-Lys transport V(max) in the PLC did not differ (121 ± 26 and 180 ± 26 pmol•mg of protein(-1)•s(-1), respectively; P = 0.14) and l-Lys K(M) in the PLC tended to be greater (0.23 ± 0.22 and 0.89 ± 0.22 mM, respectively; P = 0.09). In the pony, compared with the DJ, l-Lys transport V(max) in the PLC was greater (62 ± 25 and 149 ± 25 pmol•mg of protein(-1)•s(-1), respectively; P = 0.04) and l-Lys K(M) in the PLC was greater (0.08 ± 0.22 and 1.05 ± 0.22 mM, respectively; P = 0.02). l-Lysine diffusion was not different between segments; however, total intestinal diffusion was greater (P = 0.03) in the pony than in the pig (115 ± 10 and 73 ± 10 pmol·mg of protein(-1)•s(-1), respectively). These results demonstrate that the large colon is capable of l-Lys transport across the apical epithelial membrane with greater capacity and less affinity than the jejunum, indicating that the large colon may play a significant role in l-Lys absorption and homeostasis in hindgut fermenters.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Sodium-Hydrogen Exchanger Regulatory Factor 1 (NHERF-1) Transduces Signals That Mediate Dopamine Inhibition of Sodium-Phosphate Co-transport in Mouse Kidney

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.

Different Alterations in Rat Intestinal Glutamine Transport during the Progression of CLP- and LPS-induced Sepsis

A marked deficiency of glutamine in clinical critical illness is correlated with mortality in the intensive care unit. Though intestinal glutamine transport was reported to be impaired in late sepsis, we hypothesized that there might be a different alteration in the early stage, with differential effects on the Na(+)-dependent glutamine transporters B(0)AT1, ATB(0,+), and ATA2. Sepsis was induced by cecal ligation and puncture or lipopolysaccharide intraperitoneal injection in Sprague Dawley rats, and the samples were collected at 0, 2, 6, 12, 24h. Small intestinal brush border glutamine transport was studied by a rapid filtration technique. The relative contributions of the three main transporter, B(0)AT1, ATB(0,+), and ATA2, were determined by competitive inhibition. The mRNA level of each transporter was analyzed by RT-PCR, and an extra immunohistochemistry analysis was performed to detect the localization of ATA2 protein in small intestine. Serum TNF-α and IL-10 concentrations were quantitated by ELISA. Intestinal glutamine transport showed a biphasic change with an early increase and a late decrease in both CLP and LPS group. The early increase of glutamine transport was mainly attributable to the increased contributions of ATA2 and ATB(0,+). The transport activities of B(0)AT1, ATB(0,+) altered mainly because of the number of transporters (mRNA level as an indicator), while turned to ATA2, the redistribution was also found to be involved. The plasma TNF-α and IL-10 levels, especially the former, showed similar changing profiles to glutamine transport and, thus, may have relevance to it. Rat intestinal glutamine transport showed an early increase and a late decrease in sepsis, and may provide some information for sepsis treatment.

Inhibitory Effects of Terpenoids on Multidrug Resistance-Associated Protein 2- and Breast Cancer Resistance Protein-Mediated Transport

The possibility of interactions between natural products/supplements and conventional prescription medicines is one of the most important issues in pharmacotherapeutic safety. Recently, we reported that some terpenoids such as (R)-(+)-citronellal and glycyrrhetic acid, which are present in herbal medicines, can act as inhibitors of P-glycoprotein (MDR1/ABCB1). In the present study, the effects of seven terpenoids on multidrug resistance-associated protein 2 (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2)-mediated transport were investigated in vitro. Membrane vesicles were prepared from MRP2 cDNA transfected Sf9 cells derived from pupal ovarian tissue of Spodoptera frugiperda, a fall armyworm, and BCRP cDNA transfected LLC-PK1 cells derived from porcine kidney. MRP2- or BCRP-mediated efflux transport was measured as ATP-dependent accumulation of [(3)H]estradiol 17-beta-d-glucuronide (E(2)17betaG) into membrane vesicles collected by a rapid filtration technique. The effects of (R)-(+)-citronellal, (S)-(-)-beta-citronellol, alpha-terpinene, terpinolene, (-)-beta-pinene, abietic acid, and glycyrrhetic acid on the intravesicular accumulation of [(3)H]E(2)17betaG were examined. Large decreases in the [(3)H]E(2)17betaG accumulation into vesicles from MRP2-overexpressing Sf9 cells were observed in the presence of glycyrrhetic acid and abietic acid, and their IC(50) values were about 20 and 51 microM, respectively. [(3)H]E(2)17betaG accumulation into vesicles from BCRP-overexpressing LLC-PK1 cells was suppressed by only glycyrrhetic acid, with an IC(50) value of about 39 microM. Other terpenoids used in this study did not alter the ATP-dependent accumulation of [(3)H]E(2)17betaG. These findings suggest that glycyrrhetic acid and abietic acid can potently inhibit MRP2- or BCRP-mediated membrane transport and may interact with their substrates in pharmacokinetic processes.

Adaptation of Epithelial Sodium-Dependent Phosphate Transport in Jejunum and Kidney of Hens to Variations in Dietary Phosphorus Intake

The objective of this study was to explore the homeostatic response of jejunal and renal epithelia regarding the inorganic phosphate (Pi) transport capacities to variations in dietary total phosphorus (tP) supply in hens. Adaptive processes were determined by quantitative measures of intake and excretion, Pi transport studies across brush border membranes, and semiquantitative detection of sodium-dependent phosphate transporters (NaPi II) based on mRNA expression in the jejunum and kidney. Twelve hens (4/group) were adapted to 3 tP feeding levels in a pair-fed manner (60 g/d): low P diet with 0.073% tP, medium P diet with 0.204% tP, and high P diet with 0.343% tP. Excretion was measured during the last 5 d of a 16-d feeding period. After slaughtering, jejunal mucosa and renal cortex were removed. Tissues were used for 32 P uptake studies in brush-border membrane vesicles by rapid filtration technique and NaPi II mRNA expression studies by northern analyses. Plasma Pi concentrations were additionally measured. The NaPi II transporter mRNA could specifically be detected in chicken jejunum and kidney. Functional parameters of Na+-dependent Pi transport indicated that these transporters were involved in chicken Pi transport across the apical membranes of jejunal and renal epithelia. Increased tP intake resulted in an increased overall tP excretion. Correlating individual data from all animals by linear regression highlighted that the adaptive decrease of renal Pi transport capacity and NaPi IIa mRNA expression was associated with an increase in plasma Pi levels and resulted in a higher tP excretion. Jejunal Pi transport capacity and NaPi IIb mRNA expression did not react to variations in dietary tP supply. In conclusion, the homeostatic response was mainly based on the adaptive capacity of the kidney in hens.

Sodium-dependent phosphate transport across the apical membrane of alveolar epithelium in caprine mammary gland

NaPi IIb cotransporter is expressed in various tissues including mammary glands of mice. The physiological role of NaPi IIb in lactating mammary glands is still unclear. Therefore, it was the aim of the study to detect and to localize NaPi IIb protein in lactating goat mammary glands by Western analysis and immunohistochemistry. Furthermore, Na(+)-dependent P(i) uptake into apical membrane vesicles isolated from goat milk was determined using rapid filtration technique. NaPi IIb protein could specifically be detected in the apical membranes of lactating alveolar epithelial cells. Na(+)-dependent P(i) uptake into apical membrane vesicles could be measured, which was inhibited by phosphonoformic acid. The kinetic parameters were V(max) with 0.9 nmol/mg protein/10 s and K(m) with 0.22 mmol/L for P(i) affinity, K(m) value for Na(+) affinity 11 mmol/L. Stoichiometry of this mammary gland Na(+)/P(i) transport across the apical membranes seemed to be 1:1 P(i):Na(+) without cooperativity in P(i) and Na(+) binding as assessed by Scatchard and Hill plots. These features of Na(+)/P(i) transport suggest that it could be mediated by NaPi IIb. The quantitative role of this P(i) transport which is directed from the alveolar lumen into the epithelial cell of goat mammary gland will be the topic of further investigations.

Isolation of Intestinal Brush‐Border Membranes

This unit provides protocols for isolating intestinal brush-border membranes from rat, pig, and cow. These membranes can be used for immunoblotting or other analytical techniques. They will also spontaneously form closed vesicles which allow for flux assays to be performed using rapid filtration techniques. Overall the isolation procedures take approximately 3.5 hr. The resulting isolated membranes can be stored under liquid nitrogen or at -70 degrees C for a week or more depending upon the species.

Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing

Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing

GSH Inhibits Trypsinization of the C-terminal Half of Human MRP1

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.

Direct Effect of CoCl2 and NiCl2 on Citrate Uptake by the Rat Renal Brush Border Membrane

Co and Ni are essential but relatively rare elements as to organisms. In the mammalian membrane, these metals are transported by the same carrier proteins. The aim of this study was to investigate the direct effects of CoCl2 and NiCl2 on citrate uptake by rat renal brush border membrane vesicles (BBMV). BBMV were prepared by the divalent cation precipitation methods, and citrate uptake was measured by the Millipore rapid membrane filtration technique. The time course of citrate uptake during 120-min of incubation with 1 mM CoCl2 and NiCl2 showed a rapid significant inhibition at the early phase and a slight recover at the late phase. Incubation for 1 min of BBMV with 1, 5 and 25 mM CoCl2 and NiCl2, respectively, significantly inhibited citrate uptake in a concentration-dependent manner compared with that of 0 mM. We discuss these findings from the point of view that Co and Ni are located in Group VIII of the periodic table.

Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and P(i). In rat, biliary P(i) concentration (0.01 mM) is approximately 100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary P(i). We aimed to functionally characterize canalicular P(i) reabsorption in rat liver and to identify the involved P(i) transport system(s). P(i) transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative P(i) transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent P(i) uptake. Initial P(i) uptake rates were saturable with increasing P(i) concentrations, exhibiting an apparent K(m) value of approximately 11 muM. P(i) transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of P(i) from primary hepatic bile and thus might play an important role in the regulation of biliary P(i) concentration.

Rapid and Slow Sand Filtration Techniques and Their Efficacy at Filtering Triactinomyxons of Myxobolus cerebralis from Contaminated Water

AbstractSlow and rapid sand filtration methods were tested for their ability to remove triactinomyxon actinospores (Tams), the waterborne infective stage of the salmonid parasite Myxobolus cerebralis, from contaminated water. Within the rapid sand filtration treatments, two backflush protocols were tested. The first consisted of extended backflush duration, and the second consisted of diverted flow past the aquaria with fish for 5 min after backflushing. A slow sand filter treatment served as a nonbackflushed control to the two rapid sand filters and also as its own unique filtration technique. Negative and positive controls were run simultaneously and served both slow and rapid sand filters. The sand used consisted only of particles greater than 180 μm (diameter). Triactinomyxon actinospores were regularly introduced to the fish‐rearing systems above the sand filters. After 60 d, clinical signs of whirling behavior and black tails were seen among the positive controls. A polymerase chain reaction (PCR) assay for M. cerebralis conducted at the study's conclusion indicated no infection among the negative controls and both of the rapid sand filter treatments. In the slow sand filter treatment 1.6% of all fish were infected, whereas 98% of the positive controls were infected. Portions of the same tissue samples used for the PCR analysis were also assayed according to the pepsin–trypsin digest (PTD) test. Within the rapid sand filters, 2.9% of fish within the long back flush treatment were infected, as were 100% of the positive controls. The diverted backflush, slow sand filter, and negative controls were all negative according to the PTD test. These results demonstrate that the backflush technique is important in the proper function of rapid sand filters used to remove Tams and that both rapid and slow sand filtration could be viable options in treating hatchery water supplies that are contaminated with whirling disease.