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The gasification behavior of six different biomass species (four forest pruning residues including Pinus pinaster, Pinyon pine, Eucalyptus, and white poplar as well as two types of garden prunings) has been investigated in a pilot fluidized bed gasifier to experimentally quantify (i) the yields of pyrolytic gas released from the fuel particle in the absence of oxygen; (ii) the effect of oxygen on the gas yields and the significance of the secondary reactions (gasification of char and reforming/cracking of gas species) during gasification with air; and (iii) the effect of fuel throughput on gasification performance. Continuous steady-state experiments were conducted using air at temperatures 800, 850, and 900 °C, equivalence ratios (ER) in the range of 0.16–0.32, and throughputs between 245 and 426 kg/h/m2, as well as semicontinuous (batch of fuel in a continuous gas stream) devolatilization experiments with N2(ER = 0). Although significant quantitative differences in the gas yields, char conversion, gas heating value, and gasification efficiency were found for different fuels, the trends with changing temperature and ER followed similar characteristics. P. pinastergave the best results, showing cold gas efficiencies of up to 70% at 900 °C, while white poplar and garden prunings performed considerably worse. The effect of throughput was analyzed for three out of six fuels, and it was observed to be small for P. pinasterand garden prunings, whereas it was significant for white poplar as a result of the lower bulk density of white poplar compared to the other fuels. The results conclude about gas composition and process efficiency expected from different biomass residues in fluidized bed gasifiers and provide fundamental data to validate a theoretical model, currently under development, to scale up the process.

Summary.: In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates.

Abdominal scintigraphy shows silent gut inflammation in patients with spondyloarthropathies (Sp) without clinical evidence of gut inflammation. Abdominal scintigraphy images are different than those obtained in patients with ulcerative colitis or Crohn's disease and are not related to the anti-inflammatory drugs administered. The aim of this study was to examine the clinical associations of findings on abdominal scintigraphy in patients with Sp. A total of 204 Sp patients (European Spondylarthropathy Study Group 1991 criteria) and 54 non-Sp controls receiving non-steroidal anti-inflammatory drugs were studied. Abdominal scintigraphy images were obtained at 30 and 120 min after injection of technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO)-labelled leucocytes. 99mTc-HMPAO-labelled leucocyte scans were positive in 104 Sp patients (50.9%) and in six non-Sp controls (2.9%) (P<0.001; OR=8.32; 95% CI=3.23–22.67). Silent gut inflammation was not associated with any of the following: age of onset, duration of evolution, sex, family history of Sp or psoriasis, articular manifestations, extra-articular manifestations, radiological findings or HLA-B27 positivity. Positive abdominal scintigraphy was associated with active disease (P<0.0001; OR=52.7; 95% CI=19–145.6) and an increase in the C-reactive protein (P<0.005; OR=3.4; 95% CI=1.5–7.4). It is concluded that (a) abdominal scintigraphy using 99mTc-HMPAO-labelled leucocytes is of value in detecting the silent gut inflammation in Sp patients, and (b) silent gut inflammation is related to the clinical activity, but is not associated with any particular type of illness or with HLA-B27.

The bile canalicular membrane contains several ATP-dependent transporters that are involved in biliary secretion. Canalicular transporters are synthesized in ER, modified in Golgi and transported to the apical plasma membrane. However, the route and regulation of intracellular trafficking of ATP-dependent transporters have not been elucidated. In the present study, we generated a translational fusion of mdr1 and green fluorescent protein and investigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line. Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i.e. rhodamine-123) into the bile canaliculi. Canalicular secretion of fluorescein isothiocyanate-glycocholate was stimulated by taurocholate and a decapeptide activator of phosphoinositide 3-kinase and was decreased by wortmannin. WIF-B9 cells were transiently and stably transfected with a mdr1-GFP construct. Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majority of which traveled directly to the canalicular membrane. Recycling between the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basolateral plasma membrane. At 15 degrees C, mdr1-GFP accumulated in Golgi; after a shift to 37 degrees C, fluorescence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin. In these studies as well, no mdr1-GFP fluorescence was observed at any time in basolateral membranes or other intracellular organelles. In conclusion, in WIF-B cells, there is a direct route from Golgi to the canalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependent transporter of organic cations. As in normal hepatocytes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells. WIF-B cells stably transfected with mdr1-GFP provide an important model in which to study trafficking and regulation of canalicular transporters. Movies available on-line: http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html

Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking. We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATP-dependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions. Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system. Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs. These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.

Ligandin is a major renal organic anion-binding protein and is the major glutathione (GSH)-S-transferase in the rat, monkey, and man. GSH transferase activity was measured in isolated segments of the normal rabbit nephron using 1-chloro-2,4-dinitrobenzene as substrate. Enzyme activity was confined to the proximal convoluted and proximal straight tubules and was not detectable in the loops of Henle and collecting tubules.Incubation of proximal tubules with [14C]ethacrynic acid resulted in formation of glutathione and cysteine adducts. Loops of Henle and collecting tubules formed significantly less glutathione adduct and no other adducts. GSH transferase in the proximal tubule may thus be important in vivoin the formation of thiolated adducts by the kidney.

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane.

Ligandin (Y protein) is an abundant cytoplasmic glutathione transferase present in liver, kidney and gut in various animals and man. Its interaction with four radiologic contrast media (Telepaque, 3-(3 amino-2,4,6, triiodophenyl -2 ethylpropanoic acid, sodium salt; Hypaque, sodium -3, 5-diacetamido-2,4,6,-triiodobenzoate; Cholografin, N,N'adipyl-bis-(3-amino-2,4,6-triiodobenzoic acid) Nmethyl-glucosamine; Diodrast, 3,5-Diiodo-4-pyridone-N-acetic acid, Diethanolamine Salt was investigated by observing inhibitory effects on the enzymecatalyzed conjugation of glutathione with 1-chloro-2, 4-dinitrobenzene. lineweaver-Burk plots of reciprocal initial velocity versus reciprocal inhibitor concentrations at fixed glutathione and chlorodinitrobenzene concentrations demonstrate non-competitive inhibition by all contrast media except Diodrast. No conjugates of contrast media with glutathione were formed. It is postulated that intracellular accumulation of contrast media is aided by intracellular binding with ligandin. Inhibition of the GSH transferase activity of ligandin can disrupt the mercapturate formation, an important detoxification process.

When dormant hazel seeds were subjected to six weeks chilling at 5° C their subsequent transfer to 20° C resulted in the accumulation of gibberellin (GA) followed by germination. In the presence of either phosphon D or ß-chlorethyltrimethylammonium chloride (CCC) at 20° C there was inhibition of both GA accumulation and germination, a finding consistent with the hypothesis that GA biosynthesis is a necessary prerequisite for the germination of chilled hazel seeds. As abscisic acid showed a strong inhibition of germination but had little effect on GA accumulation it is presumed not to have affected GA biosynthesis but to have inhibited GA action. These conclusions were supported by experiments in which the interaction of exogenous GA3 with growth retardants and ABA was tested on the germination of chilled hazel seeds. Experiments in which the embryonic axes and cotyledons of chilled seeds were incubated separately at 20° C established that GA biosynthesis de novo occurred in the embryonic axis and indicated that in the intact seed some of the GA would have been translocated to the cotyledons. The isolated cytoledons showed no GA biosynthesis de novo but gave some release of GA from one or more bound forms.

Conclusions: The initial discovery of p-glycoprotein in the plasma membrane of MDR cancer cell lines was followed quickly by the cloning of its gene. Sequence analysis of cloned cDNAs revealed that p-glycoprotein was a member of the ABC family of membrane transporters. Subsequent biochemical characterization demonstrated the binding of chemotherapeutic drugs and ATP to p-glycoprotein. P-glycoprotein-mediated drag transport and drug-stimulated ATPase activity were documented in plasma membrane vesicles and in proteoliposomes containing the partially purified protein. P-glycoprotein was shown to be phosphorylated and the effect of this modification on the protein's biological function was explored. P-glycoproteins were found in many normal tissues and their overexpression was documented in numerous cancers. An important role for p-glycoprotein in intrinsic and acquired drug resistance in clinical oncology was established. Despite all that has been learned about p-glycoprotein over the last few years, additional studies will be necessary to address the many questions that have been left unanswered. Determination of p-glycoprotein structure and membrane topology should help elucidate the nature of chemotherapeutic drug binding sites and the mechanism whereby drug movement is coupled to ATP hydrolysis. Complete purification and functional reconstitution of p-glycoprotein into defined lipid vesicles will permit further characterization of drug transport and ATPase activity and give us the means by which p-glycoprotein's apparent dual function as a transporter and a channel can be clarified. Structural and functional studies on p-glycoprotein will also provide information needed to develop specified inhibitors that can be used clinically to overcome MDR in cancer patients. Further study of the mechanisms whereby p-glycoprotein expression is induced and regulated during malignant transformation is indicated. The development of biliary phospholipid deficiency in mdr2 knockout mice and xenobiotic hypersensitivity in mdr3 knockout mice have given us the first clues into the normal physiologic roles for the p-glycoproteins. The search for endogenous substrates for the p-glycoproteins will continue to be an area of active investigation.Continued investigation of p-glycoprotein's functions should result in better understanding of an important class of prokaryotic and eukaryotic membrane transporters. The potential of exploiting the knowledge garnered from these studies in the treatment of neoplastic, parasitic and inherited and acquired liver disease may be greater than we can now imagine.

ATP-dependent transport of bile acids is a key determinant of bile flow in mammalian liver and is associated with cholesterol excretion, gallstone formation, and numerous inherited and acquired hepatobiliary diseases. Secretory vesicles and a vacuole enriched fraction purified from Saccharomyces cerevisiae also exhibit ATP-dependent bile acid transport. ATP-dependent transport of bile acids by the vacuolar fraction was independent of the vacuolar proton ATPase, responded to changes in the osmotically sensitive intravesicular space, and was saturable, exhibiting a Km of 63 microM for taurocholate. The BAT1 (bile acid transporter) gene was isolated from yeast DNA by polymerase chain reaction amplification using degenerate oligonucleotides hybridizing to conserved regions of ABC-type proteins. ATP-dependent bile acid transport was abolished when the BAT1 coding region was deleted from the genome and restored upon reintroduction of the gene. The deduced amino acid sequence predicts that Bat1p is an ABC-type protein 1661 amino acids in length, similar to mammalian cMOAT/cMRP1 and MRP1 transporters, yeast Ycf1p, and two yeast proteins of unknown function. Information obtained from the yeast BAT1 gene may aid identification of the gene encoding the mammalian bile acid transporter.

Secretory mutants (sec1, sec6) of Saccharomyces cerevisiae accumulate large pools of secretory vesicles at the restrictive temperature (37 degrees C) because of a block in the delivery of vesicles to the cell surface. We report that secretory vesicles isolated from sec mutants exhibit ATP-dependent uptake of two classes of organic anions that are substrates for the canalicular carriers of mammalian liver. Transport of the bile acid taurocholate (TC) and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene (GS-DNP) into vesicles was temperature dependent and saturable and required ATP and Mg2+. Estimates of Km and Vmax were 177 microM and 1.2 nmol.min-1.mg-1 and 262 microM and 0.53 nmol.min-1.mg-1 for TC and GS-DNP, respectively. TC and GS-DNP did not complete for transport. TC transport was sensitive to vanadate and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited by glycocholate, and retained partial activity when UTP and GTP, but not nonhydrolyzable ATP analogues, replaced ATP. Dissipation of the electrochemical potential with a nitrate buffer and ionophores partially decreased (30-40%) the transport of both anions. Direct testing of the influence of membrane potential was performed in sec6-4 mutants, in which the expression of electrogenic [H+]ATPase activity is reduced by > 85% in glucose-containing medium. Vesicles from sec6-4 retained full activity for ATP-dependent TC and GS-DNP transport. These results indicate that the transporters operate independently of the membrane potential and that ATP is required. These findings reveal that yeast possess separate ATP-dependent transport mechanisms for elimination of bile acids and glutathione conjugates. The mechanisms are functionally similar to those present in mammalian systems.

Nonlinear chirped pulse compression can be theoretically achieved to any order by using a nonplane grating with adequate groove spacing. We evaluate the holographic recording of a grating that compensates to the quadratic chirp. A suitable design is found, and the building tolerances are analyzed.

Physiological properties of isolated pairs of rat hepatocytes were examined within 5 h after dissociation. These cells become round when separated, but cell pairs still display membrane specializations. Most notably, canaliculi are often present at appositional membranes which are flanked by abundant gap and tight junctions. These cell pairs are strongly dye-coupled; Lucifer Yellow CH injected into one cell rapidly diffuses to the other. Pairs of hepatocytes are closely coupled electrically. Conductance of the junctional membrane is not voltage sensitive: voltage clamp studies demonstrate that gj is constant in response to long (5 s) transjunctional voltage steps of either polarity (to greater than +/- 40 mV from rest). Junctional conductance (gj) between hepatocyte pairs is reduced by exposure to octanol (0.1 mM) and by intracellular acidification. Normal intracellular pH (pHi), measured with a liquid ion exchange microelectrode, was generally 7.1-7.4, and superfusion with saline equilibrated with 100% CO2 reduced pHi to 6.0-6.5. In the pHi range 7.5-6.6, gj was constant. Below pH 6.6, gj steeply decreased and at 6.1 coupling was undetectable. pHi recovered when cells were rinsed with normal saline; in most cases gj recovered in parallel so that gj values were similar for pHs obtained during acidification or recovery. The low apparent pK and very steep pHi-gj relation of the liver gap junction contrast with higher pKs and more gradually rising curves in other tissues. If H+ ions act directly on the junctional molecules, the channels that are presumably homologous in different tissues must differ with respect to reactive sites or their environment.

Three cationic glutathione S-transferase forms isolated from rat liver were characterized as dimers that originated from different combinations of two subunit types, Ya and Yc. The cationic forms were purified using lysyl glutathione affinity matrices and were chromatographically resolved from anionic glutathione S-transferases that contain Yb subunits. The three classes of cationic transferase exhibited similar specific activities with 1-chloro-2,4-dinitrobenzene as a substrate, all forms cross-reacted with antibodies to glutathione S-transferase B, and all had comparable secondary structures and tryptophan fluorescence properties. In spite of those similarities, the Yc-containing forms were clearly distinguishable from Ya forms on the basis of characteristic differences in circular dichroic patterns associated with their aromatic side chains. All cationic transferases bound bilirubin with stoichiometric ratios of 1 mol/dimeric protein molecule, but discrete differences in mode of binding were ascribed to forms containing Ya subunits as compared to Yc dimers. Binding to Yc forms was of lower affinity and may be associated with the catalytic region of the protein since glutathione effectively displaced bilirubin from the Yc component.

In this work we demonstrate the appearance of a pulse crest curvature which differs from wave-front curvature in certain chirped pulse compressors within the femtosecond range. This happens because the spectrum is made to focus on a grating or a holographic optical element, and this cannot be achieved for all wavelengths. The phenomenon is analysed from the viewpoint of geometric diffraction, as a function of chromatism, and is tested by ray tracing.

The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature-dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side-out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP-dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders.

TR- mutant Wistar rats secrete markedly fewer organic anions other than bile acids from the liver into the bile than do control rats. Fluorescence-image analysis of isolated normal and TR- hepatocyte "doublets", which retain a bile canaliculus between them, revealed that normal hepatocytes readily transport a fluorescent bile acid (fluorescein isothiocyanate glycocholate) and a nonbile acid organic anion (carboxydichlorofluorescein diacetate) into the canaliculus. Hepatocyte doublets from TR- rats also transported fluorescein isothiocyanate glycocholate normally, but transport of carboxydichlorofluorescein diacetate into the canaliculus was negligible. Vesicles derived from the canicular domain of the plasma membrane of hepatocytes (CMV) from control and TR- rats were used to characterize the transport process for 35S-labeled bromosulphthalein and 35S-labeled bromosulphthalein glutathione, which represent nonbile acid organic anions. CMV from normal rat hepatocytes had an ATP- and temperature-dependent, saturable transport process for these 35S-labeled compounds that was absent in CMV from TR- rats. CMV from TR- rats retained normal ATP-dependent transport of daunomycin, and immunologic blots with a monoclonal antibody against the multidrug resistance gene product, P-glycoprotein, revealed no difference between normal and TR-CMV. These studies reveal that the bile canaliculus in normal rats contains an ATP-dependent organic anion transport system that is functionally absent in TR- mutant rats. The defect in TR- mutant rats is phenotypically similar to that seen in mutant Corriedale sheep and in the Dubin-Johnson syndrome in man.

Circular dichroism methods were used to detect bilirubin-ligandin interactions in rat liver cytosol and fractions obtained at various stages during purification of ligandin. Ligandin retained its capacity to bind bilirubin in the presence of components of liver supernatant, but albumin, which binds bilirubin in serum, lost the capacity to bind bilirubin in liver supernatant. This was attributed to a greater binding specificity exhibited by ligandin. In their respective physiological milieus, albumin and ligandin are structurally adapted to bind ligands: albumin in serum, and ligandin in the cytosol of the liver cell. These studies are consistent with the hypothesis that the concentration of ligandin within the liver could regulate the net flux of certain organic anions from plasma into the liver.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct ‘maps’, indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.

On routine blood screens, persistent conjugated hyperbilirubinemia was discovered in two groups of closely related adult female golden lion tamarins (Leontopithecus rosalia rosalia, n = 8). Bromosulfophthalein (BSP) retention tests were performed on four hyperbilirubinemic and three control tamarins. BSP excretion was delayed in hyperbilirubinemic tamarins as compared with controls. Grossly, liver of affected tamarins was dark brown to black, with a prominent reticulated pattern. Histologic examination revealed abundant intrahepatic pigment, primarily in a centrilobular and midzonal distribution. Most of the pigment did not react with Perls' Prussian blue method for iron. Hall's method for bilirubin, or the Armed Forces Institute of Pathology acid-fast method for lipofuscin but was positive with Fontana and lipofuscin-ferric ferricyanide reduction techniques. Liver from control golden lion tamarins had intrahepatocellular Perls' iron-positive pigment diffusely throughout the lobule with a small amount of Fontana method-positive pigment. Ultrastructurally, hepatocytes from a hyperbilirubinemic tamarin contained pleomorphic electron-dense structures within lysosomes. Transport studies demonstrated secretion of fluorescein isothiocyanate-labeled glycocholic acid, a fluorescent bile acid analog, into bile canaliculi and no secretion of carboxydichlorofluorescein diacetate, a non-bile acid organic anion, by liver from a hyperbilirubinemic tamarin. In contrast, control liver secreted carboxydichlorofluorescein diacetate readily into bile canaliculi. The clinicopathologic presentation of this syndrome in golden lion tamarins is similar to that described for Dubin-Johnson syndrome of human beings.

The regulation of the inositol 1,4,5-trisphosphate (IP3) receptor in liver was analyzed using a novel superfusion method. Hepatic microsomes were loaded with 45Ca2+, and superfused at high flow rates to provide precise control over IP3 and Ca2+ concentrations ([Ca2+]) and to isolate 45Ca2+ release from reuptake. 45Ca2+ release was dependent on both [Ca2+] and IP3. The initial rate of 45Ca2+ release was a biphasic function of [Ca2+], increasing as [Ca2+] approached 3 microM but decreasing at higher concentrations, suggesting that the hepatic IP3 receptor is regulated by [Ca2+] at two sites, a high affinity potentiation site and a low affinity inhibitory site. The relationship between initial rates and IP3 concentration was steep (Hill coefficient of 3.4), suggesting that activation of the calcium channel requires binding of at least 3 IP3 molecules. IP3 concentrations above 10 microM produced rapid decay of release rates, suggesting receptor inactivation. Superfusion with 10 microM IP3 under conditions that minimize calcium release ([Ca2+] < 1 nM) inhibited 45Ca2+ release in response to subsequent stimulation (400 nM Ca2+). These data suggest sequential positive and negative regulation of the hepatic IP3 receptor by cytosolic calcium and by IP3, which may underlie hepatocellular propagation of regenerative, oscillatory calcium signals.

A proliferation-competent adult rat liver cell monolayer system has been analyzed for tissue-specific functions during its growth cycle. High levels of the adult (L type) form of pyruvate kinase (EC 2.7.1.40) and glutathione S-transferase B ("ligand," EC 2.5.1.18) are observed during the early lag phase; they decline markedly during the logarithmic phase and reappear during the stationary phase. By contrast, elevated levels of the fetal (K type) form of pyruvate kinase and alpha1-fetoprotein production appear only after proliferation begins; this pattern diminishes slightly during stationary phase as the adult phenotype is restored. Albumin production continues throughout the entire growth cycle. These in vitro findings simulate those observed during hepatoproliferative transitions in the intact animal and, as such, constitute a developmental program for normal epithelial cells in primary culture.

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)

In this paper we describe a simple method of characterizing the efficiency of bleaching holographic gratings by means of the measurement of the index image for zero frequency. It is applied to the bleaching method by immersion in a bromide atmosphere.

We previously characterized a purine-specific Na(+)-nucleoside cotransport system in bile canalicular membrane. The function of this transport system may be related to conserving nucleosides and preventing cholestasis. We report here the isolation of a cDNA encoding a Na(+)-dependent nucleoside transporter from rat liver using an expression cloning strategy. The substrate specificities and kinetic characteristics of the cloned cotransporter are consistent with the properties of the Na(+)-dependent, purine-selective nucleoside transporter in bile canalicular membranes. The nucleotide sequence predicts a protein of 659 amino acids (72 kDa) with 14 putative membrane-spanning domains. Northern blot analysis showed that the transcripts are present in liver and several other tissues. Data base searches indicate significant sequence similarity to the pyrimidine-selective nucleoside transporter (cNT1) of rat jejunum. Although these two subtypes of Na(+)-nucleoside cotransporter have different substrate specificities and tissue localizations, they are members of a single gene family.

Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.

Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

1. These studies confirm the observations of Inscoe and Axelrod that 3,4 benzpyrene administration to newborn rats enhances hepatic glucuronide formation. 2. Administration of chlorcyclizine or chloroquine to rats in late pregnancy increased glucuronide formation by homogenates of neonatal rat liver. 3. In rats and humans, chloroquine crosses the placenta and in rats chloroquine is concentrated in maternal and fetal liver. Administration of chloroquine, in the doses used to suppress malaria, to women in late pregnancy was without effect on the duration and magnitude of the hyperbilirubinemia observed in their infants. Further studies of the mechanism of these drug effects and their possible application to man are indicated.

1. Eight rats developed detectable glutathione-S-transferase activity in their urine after three daily injections of toxic doses of gentamicin. 2. Seven of the eight rats had immunodetectable ligandin in their urine at this time. 3. The level of enzyme activity correlated well with the degree of elevation of serum creatinine. 4. This confirms ligandinuria and urinary glutathione-S-transferase as markers of acute renal proximal tubular injury.

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.

THE ability of the mucosa of the gastrointestinal tract to form glucuronides has been previously reported1–3. The function of such conjugate formation in the intestinal mucosa has not yet been clarified. It has been variously suggested that glucuronide formation by this tissue may play an important part in detoxification3, in mucopolysaccharide formation4, or in the absorption of various substances from the intestinal lumen5,6. A genetically determined deficiency of the enzyme responsible for the transfer of glucuronic acid from uridine diphosphate glucuronic acid (UDPGA) to various acceptors has been detected in the livers of homozygous Gunn rats7,8. The work reported here was undertaken to determine whether there is a similar deficiency of this enzyme (UDP transglucuronylase) in the intestine of the Gunn rat. The results provide further evidence for the similarity between glucuronide synthesis in the intestinal mucosa and liver3and for the importance of glucuronide formation in the transport of various substances by rat intestine in vitro.