Mapping the local viscosity of non-Newtonian fluids flowing through disordered porous structures.

Flow of non-Newtonian fluids through topologically complex structures is ubiquitous in most biological, industrial and environmental settings. The interplay between local hydrodynamics and the fluid's constitutive law determines the distribution of flow paths. Consequently the spatial heterogeneity of the viscous resistance controls mass and solute transport from the micron to the meter scale. Examples range from oil recovery and groundwater engineering to drug delivery, filters and catalysts. Here we present a new methodology to map the spatial variation of the local viscosity of a non-Newtonian fluid flowing through a complex pore geometry. We use high resolution image velocimetry to determine local shear rates. Knowing the local shear rate in combination with a separate measurement of the fluid's constitutive law allows to quantitatively map the local viscosity at the pore scale. Our experimental results-which closely match with three-dimensional numerical simulations-demonstrate that the exponential decay of the longitudinal velocity distributions, previously observed for Newtonian fluids, is a function of the spatial heterogeneity of the local viscosity. This work sheds light on the relationship between hydraulic properties and the viscosity at the pore scale, which is of fundamental importance for predicting transport properties, mixing, and chemical reactions in many porous systems.

Hemopexin decreases spontaneous chemiluminescence of cold preserved liver after reperfusion.

Hemopexin is a plasma protein with exceptionally high affinity for heme. During liver transplantation heme is released via lysis of transfused blood. This heme may catalyze peroxidative reactions that contribute to "reperfusion" injury of the organ. Using a rat liver model of cold storage and reperfusion we tested the potential anti-oxidant effects of hemopexin. After 3 h of cold storage rat liver was reperfused with warm oxygenated buffer. Spontaneous liver chemiluminescence, which is a parameter of oxyradical production, was measured during reperfusion and expressed as an index of free radical production (IFRP). Chemiluminescence reached a maximum within 5 min of reperfusion and decreased to baseline within 30 min. Addition of hemopexin to the perfusate (5 microM) significantly decreased the IFRP. By contrast, the control proteins albumin and gamma-globulin (10 microM) had a smaller non-significant effect. The data suggest that heme could be complexed by hemopexin during reperfusion, thus inhibiting heme mediated cellular injury.

Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions.

Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37 degrees C was five- to eightfold higher than that at 4 degrees C, linear for at least 5 minutes, and saturable. The Km of heme uptake was 0.95 +/- 0.27 micromol/L, and the Vmax was 0.12 +/- 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.

The rat heme oxygenase-1 gene is transcriptionally induced via the protein kinase A signaling pathway in rat hepatocyte cultures.

Heme oxygenase-1 (HO-1) is the inducible form of the rate-limiting enzyme of heme degradation; it regulates the cellular content of heme. To investigate the role of the cAMP-dependent protein kinase (PKA) signaling pathway on hepatic HO-1 gene expression, primary rat hepatocyte cultures were treated with the PKA-stimulating agents dibutyryl-cAMP (Bt2cAMP), forskolin, and glucagon. HO-1 mRNA levels were induced by these agents in a time-dependent manner with a transient maximum after 6 hr of treatment. The induction of HO-1 was dose dependent, reaching a maximum at concentrations of 250 muM Bt2cAMP and 50 nM glucagon, respectively. The accumulation of HO-1 mRNA correlated with increased levels of HO-1 protein as determined by Western blot analysis. The Bt2cAMP-dependent induction of HO-1 mRNA expression was prevented by pretreatment with the PKA inhibitor KT5720 but not with the protein kinase G inhibitor KT5823. HO-1 mRNA induction by CdCl2 and heme was differentially affected by Bt2cAMP. Up-regulation of the HO-1 gene by Bt2cAMP occurred on the transcriptional level as determined by nuclear run-off assay and blocking of the Bt2cAMP-dependent induction of HO-1 mRNA by actinomycin D. Treatment with Bt2cAMP increased the half-life of HO-1 mRNA from 4.7 to 5.5 hr. Taken together, the results of the current study demonstrate that HO-1 gene expression is induced by activation of the cAMP signal transduction pathway via a transcriptional mechanism in primary rat hepatocyte cultures.

Hemopexin from four species inhibits the association of heme with cultured hepatoma cells or primary rat hepatocytes exhibiting a small number of species specific hemopexin receptors.

Hemopexin (Hx) binds heme with a very high affinity (Kd<0.1 pmol/L). It has been implicated as a major vehicle for the transport of heme into liver cells, involving a receptor-mediated recycling mechanism. However, previous studies indicated that heme is not taken up by cultured embryonic chick or adult rat hepatocytes by such a mechanism, because heme added as heme hemopexin failed to affect heme-responsive activities of 5-aminolevulinic acid synthase and heme oxygenase. Here, we investigated the importance of hemopexin in hepatic heme uptake in cultured rat hepatocytes and human HepG2 hepatoma cells, and determined the number and species specificity of hemopexin receptors on the rat hepatocytes. We also tested whether there is a difference between heterologous and homologous hemopexins. We found the following: 1) heme is inhibited from associating with hepatocytes by apo hemopexins from rat, human, rabbit, and chicken; 2) heme readily associates with hepatocytes when heme hemopexin preparations are added in which the ratio of heme to hemopexin exceeds 1.0; 3) heme induces heme oxygenase mRNA in rat hepatocytes and this induction is prevented by excess hemopexin; and 4) rat hepatocytes exhibit only about 2,000 hemopexin receptors per cell when using rat hemopexin, and none when using hemopexin of rabbit and human. We conclude that hemopexin plays a limited role in heme uptake by cultured hepatocytes and hepatoma cells, and that heme which exceeds the hemopexin binding capacity is taken up directly from heme-albumin.

Gene regulation of HBP 23 by metalloporphyrins and protoporphyrin IX in liver and hepatocyte cultures.

Heme-binding protein 23 kDa (HBP23) belongs to the antioxidant family of peroxiredoxins and binds heme with high affinity. In vivo treatment of rats with heme induced expression of HBP23 mRNA levels in liver coordinately with that of the heme degrading enzyme heme oxygenase-1 (HO-1). In primary rat hepatocyte cultures Sn-, Co-, and Zn-metalloprotoporphyrin as well as the heme precursor protoporphyrin IX increased the HBP23 mRNA expression to a level similar to that elicited by heme. Heme-dependent induction of HBP23 mRNA was prevented by pretreatment with actinomycin D, indicating a transcriptional mechanism of gene induction. The results suggest that the coordinate gene regulation pattern of HBP23 and HO-1 plays a physiological role against oxidative stress.

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.

Hyaluronan-binding properties of human serum hemopexin.

Hemopexin, the heme-binding serum glycoprotein, exhibits a complex electrophoretic pattern on two-dimensional immunoelectrophoresis on agarose gels into which hyaluronic acid is incorporated in the first and monospecific anti-hemopexin in the second dimension. This heterogeneity reflects a range of interactions of hemopexin isoforms with hyaluronic acid. Electrophoretic patterns of individual human sera greatly differ in their contents of hyaluronan-interacting hemopexin species. Hemopexin itself has no hyaluronidase activity.

Ferriheme and ferroheme are isosteric inhibitors of fatty acid binding to rat liver fatty acid binding protein.

In addition to fatty acids, liver fatty acid binding protein (L-FABP) also interacts with ferriheme, which it binds with an affinity approximately one order of magnitude greater than that for oleic acid. We have, therefore, examined the effect of ferroheme and ferriheme on the binding of oleate to rat L-FABP also called heme-binding protein. Both oxidation states of heme behaved as isosteric inhibitors for the binding of the fatty acid confirming a common binding site. The reduced form of heme (Fe(II)) is a threefold better competitor of oleate binding than ferriheme. To show whether the diffusion of heme would be affected by the presence of the binding protein, we measured the effect of the fatty acid binding protein on the diffusional flux of a water-soluble heme derivative, iron-deuteroporphyrin. The diffusional flux of iron-deuteroporphyrin did not change in the presence of the protein. This suggested that the binding affinity of fatty acid binding protein for iron-deuteroporphyrin is too great to allow rapid equilibrium between bound and unbound ligand across the system in an appropriate time frame.

EPR studies on the effects of complexation of heme by hemopexin upon its reactions with organic peroxides.

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.

Expression of the mRNA of heme-binding protein 23 is coordinated with that of heme oxygenase-1 by heme and heavy metals in primary rat hepatocytes and hepatoma cells.

A 23-kDa protein with high affinity for heme (KD = 55 nM), therefore termed heme-binding protein 23 kDa (HBP23), was purified from rat liver cytosol [Iwahara, S., et al. (1995) Biochemistry 34, 13398-13406]. Homology search of the cloned HBP23 cDNA revealed that this protein belongs to a recently recognized class of thiol peroxidases, the antioxidant peroxiredoxin family. Since HBP23 gene expression was highest in the liver, HBP23 mRNA regulation by heme and heavy metals was investigated in cultures of primary rat hepatocytes and mouse hepatoma Hepa 1-6 cells. In both cell cultures HBP23 mRNA levels were upregulated in a time- and dose-dependent manner by heme. Heme-dependent induction of HBP23 mRNA occurred coordinately with that of the heme-metabolizing enzyme heme oxygenase-1, which was recently identified as inducible by oxidative stress. Treatment of primary rat hepatocyte or hepatoma cell cultures with the heavy metals CdCl2 (10 microM) and CoCl2 (300 microM) induced in parallel HBP23 and HO-1 mRNA levels, in the case of CdCl2 to even higher levels than heme. By contrast, mRNA expression of another heme binding protein, hemopexin, was not induced in hepatocyte cell cultures by heme or heavy metals. The data suggest that the expression of HBP23 and HO-1 mRNA is regulated by (a) similar mechanism(s) in liver and that both genes could play a common physiological role as antioxidants and/or in heme metabolism.

Purification, characterization, and cloning of a heme-binding protein (23 kDa) in rat liver cytosol.

A heme-binding protein (designated HBP23) has been purified from rat liver cytosol using heme-affinity chromatography and either reverse-phase high-performance liquid chromatography or sequential ion-exchange chromatography. The protein (23 kDa) binds heme with an affinity (Kd = 55 nM) higher than that of the abundant cytosolic heme-binding proteins, heme-binding protein (HBP)/liver fatty acid-binding protein (L-FABP) and the glutathione S-transferases (GSTs) (Kd = 100-200 nM). HBP23 is present in the cytosol of liver, kidney, spleen, small intestine, and heart, with the liver showing the highest content. A cDNA coding the 23-kDa protein was cloned using reverse transcription polymerase chain reaction with degenerative oligonucleotides derived from partial amino acid sequences. The cloned cDNA encoded 199 amino acids, and its amino acid sequence showed no homology to HBP/L-FABP, GSTs, or any other heme-binding proteins or hemeproteins. Homology search showed that HBP23 is highly homologous to mouse macrophage 23-kDa stress protein, which is inducible by oxidant stress in peritoneal macrophages [Ishii, T., Yamada, M., Sato, H., Matsue, M., Taketani, S., Nakayama, K., Sugita, Y., and Bannai, S. (1993) J. Biol. Chem. 268, 18633-18636]. Thioredoxin peroxidase as well as HBP23 and the mouse macrophage 23-kDa stress protein are members of the peroxiredoxin family, a recently recognized class of antioxidant proteins [Chae, H. Z., Chung, S. J., & Rhee, S. G. (1994) J. Biol. Chem. 269, 27670-27678]. An increase in HBP23 mRNA was observed in Hepa 1-6 cells after treatment with heme and cadmium and during liver regeneration after partial hepatectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of hemopexin gene expression by physiological oxygen tensions in primary rat hepatocyte cultures.

Hyperoxia induces the expression of the hemopexin (Hx) gene in the liver in vivo. To investigate whether the Hx gene is activated by oxygen as such or via H2O2 as an oxygen signal transmitter the effects of arterial and venous O2 tensions as well as different concentrations of H2O2 on Hx mRNA expression were studied. After preculturing primary rat hepatocytes for 24 h at arterial O2 (16%) Hx mRNA was expressed with a maximal level (= 100%), when arterial O2 tension proceeded for 2 h, and to values of approximately 50%, when venous O2 tension (8%) proceeded for 2 h. When hepatocytes were precultured for 24 h under venous O2, Hx mRNA was induced by arterial O2 to values of 60% and under venous O2 to values of approximately 35%. The expression of beta-actin remained unchanged under arterial and venous O2. Exposure of hepatocyte cultures to H2O2 decreased the expression of Hx mRNA in a dose-dependent manner after 2 h, while heme oxygenase-1 (HO-1) mRNA was induced 2.5 fold. The results suggest that O2 per se rather than the reactive oxygen intermediate H2O2 modulates Hx expression.

Expression of haemopexin receptors by cultured human cytotrophoblast.

The expression of cell-surface haemopexin (Hx) receptors on human cytotrophoblasts was assessed by using four different Hx species purified from plasma: human Hx isolated by wheatgerm-affinity chromatography, human Hx isolated by haem-agarose-affinity chromatography and rabbit and rat Hx, also isolated by haem-agarose-affinity chromatography. About 3500-7000 high-affinity (Kd 0.34-0.85 nM) receptors per cell were measured by Scatchard-type analysis at 4 degrees C using human (species obtained by both methods) or rabbit 125I-labelled haem-Hx. Measured simultaneously, transferrin receptor number and affinity were 40,000/cell and 0.83 nM respectively. In contrast with transferrin receptors, the number of Hx receptors did not increase during 24 h in cytotrophoblast culture. Rat Hx showed no specific binding to human Hx receptors in cytotrophoblast cultures.

A gene cluster involved in the utilization of both free heme and heme:hemopexin by Haemophilus influenzae type b.

The utilization of heme bound to the serum glycoprotein hemopexin by Haemophilus influenzae type b (Hib) strain DL42 requires the presence of the 100-kDa heme:hemopexin-binding protein encoded by the hxuA gene (M. S. Hanson, S. E. Pelzel, J. Latimer, U. Muller-Eberhard, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). Nucleotide sequence analysis of a 5-kb region immediately upstream from the hxuA gene revealed the presence of two genes, designated hxuC and hxuB, which encoded outer membrane proteins. The 78-kDa HxuC protein had similarity to TonB-dependent outer membrane proteins of other organisms, whereas the 60-kDa HxuB molecule most closely resembled the ShlB protein of Serratia marcescens. A set of three isogenic Hib mutants with cat cartridges inserted individually into their hxuA, hxuB, and hxuC genes was constructed. None of these mutants could utilize heme:hemopexin. The hxuC mutant was also unable to utilize low levels of free heme, whereas both the hxuA and hxuB mutants could utilize free heme. When the wild-type hxuC gene was present in trans, the hxuC mutant regained its ability to utilize low levels of free heme but still could not utilize heme:hemopexin. The hxuA mutant could utilize heme:hemopexin when a functional hxuA gene from a nontypeable H. influenzae strain was present in trans. Complementation analysis using this cloned nontypeable H. influenzae hxuA gene also indicated that the HxuB protein likely functions in the release of soluble HxuA from the Hib cell. These studies indicate that at least two and possible three gene products are required for utilization of heme bound to hemopexin by Hib strain DL42.

The type II hemopexin interleukin-6 response element predominates the transcriptional regulation of the hemopexin acute phase responsiveness.

Hemopexin (Hx) is induced during the acute phase response (APR) by the cytokine interleukin (IL)-6. A type II IL-6 response element (RE) of the Hx gene has been characterized recently (J. Biol. Chem. (1994); 269, 12654-12661). To assess Hx gene regulation by other agents, various cytokines and growth factors were tested for their ability to induce Hx in rat hepatoma H-35 cells. IL-6-type cytokines, IL-1 beta and TNF-alpha, in contrast to transforming growth factor-beta (TGF-beta), hepatocyte growth factor and insulin significantly increased Hx gene expression. Chloramphenicol acetyltransferase (CAT) activity in H-35 cells transfected with constructs that contained the 5'-flanking Hx promoter region or multiple copies of the Hx IL-6-RE fused to the CAT gene was upregulated only by IL-6-type cytokines, although to varying degrees. These data indicate that signal transduction pathways mediated by IL-6-type cytokines but not those by IL-1 beta and TNF-alpha converge on the common Hx IL-6-RE.

Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum heme-binding protein.

Hyaluronan is the most abundant glycosaminoglycan of the extracellular matrix and is a critical substrate for cellular attachment and locomotion. Little is known about the class of enzymes, termed hyaluronidases, that are responsible for hyaluronan catabolism in mammals. We have determined a partial amino acid sequence from a purified preparation of porcine liver hyaluronidase and have used this information as the basis for cloning complementary DNA that encodes the corresponding protein. When expressed in a recombinant baculovirus system, the protein exhibited hyaluronidase activity in a substrate-gel assay. The deduced sequence of this mammalian hyaluronidase is that of a 459-amino-acid polypeptide bearing four potential N-glycosylation sites as well as a copy of a proposed hyaluronan binding motif. Remarkably, amino acid sequence comparisons and immunologic cross-reactivities strongly suggest that the cloned protein is identical to hemopexin, an abundant, heme-binding serum protein. Although hemopexin has not previously been reported to possess any enzymatic activity, it includes a conserved domain found in collagenases, stromelysins, and other enzymes that metabolize the extracellular matrix. We conclude that hemopexin is the predominant hyaluronidase expressed in mammalian liver.

Immunological identity of rat liver cytosolic heme-binding protein with purified and recombinant liver fatty acid binding protein by western blots of two-dimensional gels.

We examined the degree of similarity between rat liver cytosolic heme-binding protein (HBP) and rat liver fatty acid binding protein (L-FABP) purified from rat liver cytosol and recombinant L-FABP (rL-FABP). We compared 1) HBP, 2) L-FABP, and 3) rL-FABP prepared in three different laboratories and probed them with three different antisera also from different laboratories on Western blots. The objective was to determine whether the isoform pattern of the recombinant would resemble those of the purified rat liver proteins and whether heme is bound by the isoforms. To investigate the similarities, we compared the immunoreactivity of purified HBP, L-FABP, and rL-FABP by probing Western blots of 2-D gels with polyclonal antibodies raised against each of these proteins. All of the antibodies react with the same isoelectric species for each of the proteins. In addition, [55Fe]-heme binds equally well to the 2 major HBP isoforms.

Influence of transcriptional regulation and mRNA stability on hemopexin gene expression in regenerating liver.

The hepatic response to systemic injury is characterized by alterations in the synthesis of plasma proteins, while acute injury to the liver can lead to rapid proliferation of hepatocytes. The hemopexin gene was found to be markedly induced in rat liver following both sham surgery (SS) and 70% partial hepatectomy (PH), models of systemic injury and hepatic proliferation, respectively. Transcriptional and post-transcriptional regulation of this gene was evaluated to examine the mechanisms of hemopexin mRNA expression in these models. Significant transcriptional activation was observed within 6 h of either surgery, with a more pronounced effect after PH. In both processes, transcription rates returned to baseline values by 24 h after surgery, although marked elevations in mRNA steady-state levels were noted for at least 72 h. At each time point, levels of hemopexin mRNA were more abundant following PH than after SS, in part due to greater transcriptional induction. In addition, posttranscriptional mechanisms appeared to contribute to the increased expression of hemopexin post-PH. The in vivo half-life of the 1.6-kb hemopexin transcript was determined to be considerably greater than 12 h in control, sham-operated, and PH animals. The exceptionally long mRNA half-life appears to be an important but complex factor in the kinetics of hemopexin gene regulation.