Role of chloride and intracellular pH on the activity of the rat hepatocyte organic anion transporter.

Previous studies in cultured rat hepatocytes revealed that initial uptake of sulfobromophthalein (BSP) was markedly reduced upon removal of Cl- from the medium. In the present study, unidirectional Cl- gradients were established in short-term cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external Cl- and is not stimulated by unidirectional Cl- gradients, suggesting that BSP transport is not coupled to Cl- transport. In contrast, BSP transport is stimulated by an inside-to-outside OH- gradient, consistent with OH- exchange or H+ cotransport. As the presence of Cl- is essential for but not directly coupled to BSP transport, binding of 35S-BSP to hepatocytes was determined at 4 degrees C. This revealed an approximately 10-fold higher affinity of cells for BSP in the presence as compared to the absence of Cl- (Ka = 3.2 +/- 0.8 vs. 0.42 +/- 0.09 microM-1; P less than 0.02). Affinity of BSP for albumin was Cl(-)-independent, and was approximately 10% of its affinity for cells in the presence of Cl-. These results indicate that extracellular Cl- modulates the affinity of BSP for its hepatocyte transporter.

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

High frequency of luteal phase deficiency and anovulation in recreational women runners: blunted elevation in follicle-stimulating hormone observed during luteal-follicular transition.

The purposes of this investigation were to evaluate the characteristics of three consecutive menstrual cycles and to determine the frequency ofluteal phase deficiency (LPD) and anovulation in a sample of sedentary and moderately exercising, regularly menstruating women. For three consecutive menstrual cycles, subjects collected daily urine samples for analysis of FSH, estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and creatinine (Cr). Sedentary (n=11) and exercising (n=24) groups were similar in age (27.0+/-1.3 yr), weight (60.3+/-3.1 kg), gynecological age (13.8+/-1.2 yr), and menstrual cycle length (28.3+/-0.8 days). Menstrual cycles were classified by endocrine data as ovulatory, LPD, or anovulatory. No sedentary women (0%) had inconsistent menstrual cycle classifications from cycle to cycle, but 46% of the exercising women were inconsistent. The sample prevalence of LPD in the exercising women was 48%, and the 3-month sample incidence was 79%. In the sedentary women, 90% of all menstrual cycles were ovulatory (SedOvul; n=28), whereas in the exercising women only 45% were ovulatory (ExOvul; n=30); 43% were LPD (ExLPD; n=28), and 12% were anovulatory (ExAnov; n=8). In ExLPD cycles, the follicular phase was significantly longer (17.9+/-0.7 days), and the luteal phase was significantly shorter (8.2+/-0.5 days) compared to ExOvul (14.8+/-0.9 and 12.9+/-0.3 days) and SedOvul (15.9+/-0.6 and 12.9+/-0.4 days) cycles. Luteal phase PdG excretion was lower (P < 0.001) in ExLPD (2.9+/-0.3 microg/mg Cr) and ExAnov (0.8+/-0.1 microg/mg Cr) cycles compared to SedOvul cycles (5.0+/-0.4 microg/mg Cr). ExOvul cycles also had less (P < 0.01) PdG excretion during the luteal phase (3.7+/-0.3 microg/mg Cr) than the SedOvul cycles. E1C excretion during follicular phase days 2-5 was lower (P=0.05) in ExOvul, ExLPD, and ExAnov cycles compared to SedOvul cycles and remained lower (P < 0.02) in the ExLPD and ExAnov cycles during days 6-12. The elevation in FSH during the luteal-follicular transition was lower (P < 0.007) in ExLPD (0.7+/-0.1 ng/mg Cr) cycles compared to SedOvul and ExOvul cycles (1.0+/-0.1 and 1.1+/-0.1 ng/mg Cr, respectively). Energy balance and energy availability were lower (P < 0.05) in ExAnov cycles than in other menstrual cycle categories. The blunted elevation in FSH during the luteal-follicular transition in exercising women with LPD may explain their lower follicular estradiol levels. These alterations in FSH may act in concert with disrupted LH pulsatility as a primary and proximate factor in the high frequency of luteal phase and ovulatory disturbances in regularly menstruating, exercising women.

Interstitial microdeletion of chromosome 1p in two siblings.

Two half-siblings are described with what we believe to be the second and third cases identified of the microdeletion, del(1)(p32.1p32.3). Both siblings had a tethered cord and had mental retardation, but otherwise their phenotypic presentations were quite different. The sister had failure to thrive, congenital dysplasia of the hip, absent corpus callosum, Chiari I malformation, and syringomyelia. The brother experienced neonatal seizures secondary to a hemorrhagic stroke. He had an accessory thumb, inguinal hernia, cryptorchidism, urinary reflux, and cholelithiasis. Although these children have only delayed development in common with the previously reported case, they help to further define chromosomal deletions in this area of chromosome 1. As yet, no clear phenotypic picture has emerged.

Chlorpyrifos interferes with cell development in rat brain regions.

Chlorpyrifos, one of the most widely used pesticides, exhibits greater toxicity during development than in adulthood. We administered chlorpyrifos to neonatal rats in doses spanning the threshold for systemic toxicity and examined developing brain regions (brainstem, forebrain, cerebellum) for signs of interference with cell development using markers for cell packing density and cell number (DNA concentration and content) and cell size (protein/DNA ratio). Neonatal rats given 5 mg/kg of chlorpyrifos on postnatal days 1-4 showed significant mortality and the survivors exhibited severe cell loss in the brainstem; brainstem growth was maintained by enlargement of the remaining cells. This effect was not seen at 1 mg/kg, a dose that did not compromise survival or growth, nor was there any adverse effect at either dose in the forebrain, despite the fact that both brainstem and forebrain possess comparable cholinergic projections. When chlorpyrifos was administered later, on days 11-14, the major target for cell loss shifted from the brainstem to the forebrain and in this case, effects were seen at doses that did not compromise survival or growth. The loss of forebrain cell number occurred between 15 and 20 days of age rather than during the chlorpyrifos treatment. The cerebellum differed from the other regions in that it showed short-term elevations of DNA after chlorpyrifos exposure in either early or late postnatal periods; nevertheless, values then regressed to subnormal in parallel with the loss of cells in other regions. Thus, chlorpyrifos likely causes delayed cell death. Although regions rich in cholinergic projections, such as brainstem and forebrain, may be more affected than noncholinergic regions (cerebellum), the maturational timetable of each region (brainstem earliest, forebrain intermediate, cerebellum last) appears to be more important in setting the window of vulnerability. These results indicate that, even when growth or survival are unaffected, chlorpyrifos produces cellular deficits in the developing brain that could contribute to behavioral abnormalities.

Detailed characterization of solute transport in a heterogeneous field soil.

There is a necessity for improved physical understanding of solute transport processes in heterogeneous soil systems. In situ nondestructive techniques like time domain reflectometry (TDR) and fiber optic miniprobes (FOMPs) permit the collection of unique measurements of solute transport processes in soils for the purposes of model development and validation. This study examined the application of TDR and FOMPs to measure solute transport at various points laterally and at two depths in a heterogeneous clay-loam soil. A miscible displacement experiment was performed at a constant irrigation flux to examine the applicability of these probes to field soils. In their first application to a field soil, the FOMPs were successfully calibrated and performed well in measuring solute breakthrough curves. Two flow regimes were identified in the soil profile, the first where lateral spreading of the solute occurred in the surface horizon, followed by convergence into preferential flow pathways in the second transport zone. The measured transport response was heterogeneous with at least two identifiable vertical flow phases. It was demonstrated using transfer function modeling and data from a corresponding laboratory study that the FOMPs were measuring the slower phase, while the TDR probes captured a composite of the fast and slow phases. The combination of these two techniques may be a means to separate solute transport phases in heterogeneous media and relate laboratory column results to field studies.

Efficiency of D- vs L-methionine utilization by growing steers.

Five ruminally cannulated Holstein steers (180 kg) were used in a 5 x 5 Latin square design experiment to evaluate the efficiency of D- vs L-methionine (Met) utilization in growing steers. Treatments were continuous abomasal infusions of water or water with 2 or 4 g.steer-1.d-1 L-Met or 2 or 4 g.steer-1.d-1 D-Met. All steers were fed 2.7 kg/d (DM basis) of a soyhull-based diet to minimize basal Met flow to the small intestine to create a paradigm by which responses to Met supplementation could be measured easily. To ensure that amino acids other than Met did not limit animal performance, an essential amino acid mixture simulating the nonsulfur amino acid profile of ruminal bacteria was infused abomasally. Energy supply to the steers was increased by continuously infusing acetate, propionate, and butyrate into the rumen at rates of 150, 150, and 37.5 g.steer-1.d-1, respectively. Abomasal infusions of dextrose (300 g/d) also provided an additional energy source. Nitrogen retention increased linearly (P < .05) in response to both L-Met and D-Met infusions, with responses being similar between the two Met isomers. Regression of N retention against Met supplementation yielded: Nitrogen retention (g/d) = 19.2 + 1.81 D-Met (g/d) + 1.90 L-Met (g/d). By slope ratio analysis, the efficiency of D-Met utilization relative to L-Met was 95.5%. In a concurrent study using intestinally cannulated steers, both D- and L-Met were absorbed essentially completely from the small intestine. These results show that D-Met can be utilized efficiently as a Met source by cattle.

Sulfur amino acid utilization by growing steers.

Studies were conducted to quantify sulfur amino acid requirements of growing steers. In Exp. 1, six steers (160 kg) were used to determine the methionine requirement in the presence of excess L-Cys. Treatments were abomasal infusion of water only or water plus 2, 4, 6, 8, or 10g/d of L-Met. Steers were fed 2.4 kg/d DM of a diet designed to minimize basal Met supply to the small intestine. Continuous ruminal infusions of VFA (506 g/d) and abomasal infusions of dextrose (150 g/d) increased energy supply to the abomasum. Break-point analysis estimated maximal N retention at 5.8 g/d supplemental L-Met. The basal absorbable Met supply was 2.1 g/d; therefore, the total Met requirement was 7.9 g/d when excess Cys was available. In Exp. 2, five steers (195 kg) were used to determine the efficiency of transsulfuration. Treatments were abomasal infusion of water only or water plus 1.62 or 3.25 g/d L-Cys or 2 o4 g/d L-Met. Diet and infusions were similar to those on Exp. 1, except the amino acid mixture was devoid of Cys and all steers received 4 g/d L-Met to make total sulfur amino acids, but not necessarily Met, limiting. Nitrogen retention increased in response to Met but not Cys infusion and was maximized at 2 g/d supplemental L-Met; thus, the total Met requirement was near 8.4 g/d when the Cys supply was 2.1 g/d. Supplemental Cys did not spare Met, suggesting that nonprotein functions of Met may be quantitatively important.

Free amino acid supplementation to steers: effects on ruminal fermentation and performance.

Three studies were conducted to evaluate amino acid utilization by cattle. In Exp. 1, five steers (580 kg) were fed 86% rolled corn diets with mixtures of amino acids containing up to 6 g/d DL-Met, 24 g/d L-Lys, 6 g/d L-Thr, and 3 g/d L-Trp. Treatments had little effect on ruminal fermentation, diet digestibility, N flow to the duodenum, or microbial efficiency. Ruminal concentrations of Met and Lys increased linearly (P < .05) with amino acid supplementation, whereas Thr responded quadratically, and Trp was not altered. In Exp. 2, four steers (414 kg) were used to measure effects of dietary monensin or laidlomycin propionate in high-grain diets supplemented with amino acids. Ionophores had no significant effect on ruminal fermentation or outflows of amino acids from the rumen. In Exp. 3, 100 steers (287 kg initial BW) were fed diets containing 1% of a nonprotein N source. Treatments were 1) no supplemental N (UREA), 2) UREA plus soybean meal (SBM), 3) UREA plus 2 g/d DL-Met, 8 g/d L-Lys, 2 g/d L-Thr, and 1 g/d L-Trp, or 4) UREA plus 4 g/d DL-Met, 16 g/d L-Lys, 4 g/d L-Thr, and 2 g/d L-Trp. During the growing period (diets based on whole-plant milo silage), gains were higher for SBM-supplemented steers than for UREA steers and intermediate for steers supplemented with amino acids. Few significant differences in performance were observed among treatments during the finishing phase (diets based on dry-rolled corn) or for the entire experiment, but cattle fed SBM or amino acids tended to be fatter and have better marbling scores and quality grades. Amino acids did not greatly alter ruminal fermentation or cattle performance.

Photodocumentation reverses Medicare reimbursement denial.

The need for documentation of medical necessity is critical as payors review claims. In the case study described, because the patient exhausted benefits for in-patient treatment under Medicare, rehospitalization would have been without reimbursement. Despite her complex illness and wounds which would normally require hospitalization, the patient was presented to a home health group for interim management. Medicare, the payor in this case, initially denied payment of the claim, questioning the justification for the wound care products used. The home health agency submitted with the denial appeal the most objective documentation available, the serial photographs which clearly indicated positive outcomes. This photodocumentation led to a complete reversal of Medicare's denial and no further denial was received. Photodocumentation should be a part of a standard protocol for documentation of complex wound care.

The modified obstetric metabolic equivalent (MET): finding a MET that fits in pregnancy.

The Compendium of Physical Activities (CPA) provides the energy expenditure (EE) for hundreds of daily activities reported in metabolic equivalents (MET). It remains to be determined if the metabolic changes of pregnancy alter the use of the CPA MET (METCPA) in this population. The energy cost of rest, activities of daily living (ADL; typing, folding laundry and sweeping) and treadmill walking [2.0, 2.5, 3.0 mph (0% incline), 3.0 mph (3% incline)] were compared with the METCPA from the 2000 and 2011 CPA in 30 pregnant women (10-14 weeks gestation) using indirect calorimetry (IC). The METCPA for each activity was compared against two measured IC values: METabsolute (3.5 ml O2/kg/min) and METratio (EEactivity/EErest). Means for both comparisons were tested by one-sample t-test. Measured MET correlated with the 2011 METCPA: METabsolute v. METCPA R 2 = 0.906, P < 0.0001; METratio v. METCPA R 2 = 0.861, P < 0.0001. Differences between measured MET values and the 2011 METCPA ranged from 16% underestimation to 48% overestimation. Using the absolute definition, the METCPA significantly overestimated the ADL (P < 0.0005); yet, no significant differences were found between walking at 0% grade and METCPA. Conversely, only folding laundry was significantly different with the ratio definition, whereas walking at a level grade was significantly underestimated (P < 0.0001). Similar observations were found using the 2000 CPA. The use of the METCPA to estimate EE in pregnant women can result in significant over- or underestimation, depending on the activity and the definition of the MET that is used.

Extracellular ATP4- modulates organic anion transport by rat hepatocytes.

The hepatocyte has an organic anion transport system that recognizes compounds such as bilirubin and sulfobromophthalein. These anions circulate bound tightly to albumin from which they are extracted rapidly by hepatocytes by an electroneutral process that requires extracellular inorganic anions such as Cl- for activity. Transport activity is reduced by depletion of intracellular ATP, but whether ATP interacts directly with this transporter is not known. In this study, the influence of extracellular ATP on the hepatocyte organic anion transport mechanism has been characterized. In the presence of 2.5 mM Ca2+ and 2 mM Mg2+, initial uptake of [35S]sulfobromophthalein was reduced by 50% at 1 mM ATP. In the absence of divalent cations sensitivity to ATP was 10-fold greater. Other nucleotides including UTP, CTP, GTP, ADP, AMP, and AMP-PCP (adenosine 5'-(beta,gamma-methylene)triphosphate) were inactive. Decreased transport activity was rapidly reversible, was non-competitive with respect to ATP, did not require ATP hydrolysis, and did not correlate with P2y purinergic receptor activity. Differential activity of ATP on sulfobromophthalein transport in the presence and absence of divalent cations was not due to ecto-ATPase activity but rather to alteration in [ATP4-]. Although an ATP4- receptor in macrophages mediates increased cellular permeability, reduced organic anion permeability is seen in hepatocytes. This effect is not seen in the hepatoma cell line HepG2. Modulation of activity of the organic anion transporter by extracellular ATP may have important pathophysiological consequences in conditions resulting in liver cell injury.

Phytochemical assessment of Lathyrus species for the neurotoxic agent, beta-N-oxalyl-L-alpha-beta-diaminopropionic acid.

Lathyrus species have potential as feed crops, but many contain a neurotoxin, beta-N-oxalyl-L-alpha-beta-diaminopropionic acid (ODAP). An extraction and Spectrophotometric assay procedure are described for analysis of ODAP in small samples (0.5 g) of seed. The assay utilizes o-phthalaldehyde as a reagent, following hydrolysis of the ODAP. It is sensitive and specific, and suitable for the assay of 3-4 seeds of most Lathyrus species. The method has been used to screen a collection of Lathyrus accessions for use in a breeding program to develop agronomic strains with low toxicity.

Skeletal scintigraphy. The use of technetium 99M-labeled complexes in the detection of early osseous involvement by metastatic tumors.

Skeletal scintigraphy using Technetium-99m ((99m)Tc) complexes was carried out in a series of 332 cancer patients. The results of scintigraphy were compared with the results of roentgenography and with the diagnostic usefulness of serum alkaline and acid phosphatase levels and the presence or absence of bone pain. In 25 percent of cases, lesions were first identified with scintigraphs. When metastastic lesions were present on both scintigraphs and roentgenograms, the number was greater on scintigraphs in 72 percent of cases. Six false negative studies were recorded (1 percent). Sixty percent of patients with early metastasis-that is, those with abnormal scintigraphs and negative roentgenograms-were asymptomatic. Serum alkaline and acid phosphatase levels were normal in 40 percent and 42 percent respectively of those with early skeletal involvement. Skeletal scintigraphy with (99m)Tc complexes is superior to other commonly employed techniques used to assess bone metastasis.

Toxins in the seedlings of some varieties of grass pea (Lathyrus sativus).

The major toxin present in the dry seeds and seedlings of Lathyrus sativus is the neurotoxin 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP). The presence of one additional neurotoxin and an osteotoxin in the seedlings increases the overall toxicity. Isolation, purification, and detection of these toxins are described.

Yogurt consumption does not enhance immune function in healthy premenopausal women.

Fermented milk products may protect against breast cancer by stimulating immunologic activity. Twenty-five women [24.0 +/- 0.7 (SE) yr] were assigned randomly to two groups: control (n = 12) and yogurt treatment (n = 13). Controls refrained from yogurt products for three months, whereas the yogurt treatment group consumed two cups (454 g/day) of commercially produced yogurt for three consecutive months. Prior yogurt consumption did not exceed 4-6 cups/mo, and subjects consumed their usual diet during the study. Three-day diet records and fasting midluteal blood samples were obtained during subjects' first, second, and fourth menstrual cycles (baseline, Month 1, and Month 3, respectively). Macronutrient intakes differed between groups only for carbohydrate. Calcium intake increased for yogurt consumers during intervention. Lymphocyte proliferation induced by concanavalin A, phytohemagglutinin, and pokeweed mitogen, interleukin 2 production, and cytotoxic T lymphocyte-mediated cytotoxicity was assessed after baseline and Months 1 and 3 for both groups. No significant immune differences between the control and yogurt treatment group were observed for concanavalin A, phytohemagglutinin, pokeweed mitogen, interleukin-2, or cytotoxicity. In conclusion, three months of yogurt consumption did not enhance ex vivo cell-mediated immune function in young women.

Organic anion transport in HepG2 cells: absence of the high-affinity, chloride-dependent transporter.

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)