Differential inflammasome activation signatures following intracellular infection of human macrophages with Mycobacterium bovis BCG or Trypanosoma cruzi.

Pathogens frequently exploit or evade inflammasome activation in order to survive and proliferate. Alternatively, inadequate inflammasome activation by attenuated microorganisms or adjuvanted subunit vaccines may contribute to poor longevity of protection. To further understand these pathways, we determined the differential inflammasome transcriptome of human THP monocyte-derived macrophages in response to Mycobacterium bovis BCG, as compared to LPS or Trypanosoma cruzi. The results identify the highly specific innate recognition programs associated with inflammasome activation by human macrophages exposed to these microbial stimuli. BCG, T. cruzi, and LPS strongly induced expression of both unique and overlapping genes downstream of TLR signaling pathways including cytokines and chemokines that mediate inflammation and regulate cell death pathways. Compared to LPS, BCG failed to directly activate anti-apoptotic molecules and multiple NLR and inflammasome complex components including caspase-1, and actively repressed important signaling intermediates in AP-1 and NFκB transcription factor pathways. Both BCG and T. cruzi repressed expression of TXNIP, an anti-oxidant inhibitor that recruits caspase-1 to the NLRP3 inflammasome, while T. cruzi infection uniquely failed to activate TNF-α. These results identify unique pathogen specific strategies to activate inflammation and modulate cell death that may drive inflammatory outcomes and suggest avenues of investigation to optimize host immunity.

Medical co-morbidities of patients with haemophilia: pain, obesity and hepatitis C.

Clinical care of patients with haemophilia (PWH) has progressed rapidly over the past decade. Current therapy has allowed patients with haemophilia to live longer and many patients are now experiencing the co-morbidities of the general population. In this review article, we focus on three common diseases states that affect PWH: chronic pain, obesity and hepatitis C. Pain has been a co-morbidity for many years and PWH often have unusual needs for chronic pain relief compared to the general population. Obesity is not only increasing in the general population but also in patients with hereditary bleeding disorders. The co-morbidity of obesity not only causes increased pain progression and joint damage but also affects the dosing of factor concentrates. Finally, hepatitis C is known to have infected the majority of patients who received non-virally inactivated pooled factor concentrates in the past. New treatment regimens have been developed that allow the nearly uniform cure of chronic hepatitis C with a short course of oral medications.

Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis.

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

Rat hepatic stellate cells become retinoid unresponsive during activation.

Hepatic stellate cells (HSC) play an essential role in fibrogenesis. Many stimuli cause HSC to activate, lose their Vitamin A and produce collagen. It is unclear whether Vitamin A loss causes activation, potentiates it or is simply an event in the cascade of activation changes. We determine if exogenous retinoids prevent the activation of freshly isolated rat HSC activated by plating on plastic. We also determine if retinoids: (1) reverse HSC activation; (2) maintain/restore HSC intracellular retinoid levels; (3) maintain expression of HSC nuclear receptors for retinoic acid (RAR) in HSC that are becoming activated or are chronically activated. Markers of activation in freshly isolated HSC were decreased by either retinol or retinoic acid without increases in HSC retinoid concentration. mRNA levels for RAR-alpha, RAR-beta and RAR-gamma, the nuclear receptors for retinoic acid, decreased during activation of freshly isolated HSC even with retinoid supplementation. RAR-alpha, RAR-beta and RAR-gamma mRNA and RAR-beta protein was undetectable in chronically activated HSC and remained absent after retinoic acid supplementation. Activation markers in chronically activated HSC were only slightly decreased after retinoid exposure. We conclude that exposure of HSC to extracellular retinoids diminishes some markers of activation but does not prevent HSC activation.

Apoptin: specific killer of tumor cells?

In the early 1990s it was discovered that the VP3/Apoptin protein encoded by the Chicken Anemia virus (CAV) possesses an inherent ability to specifically kill cancer cells. Apoptin was found to be located in the cytoplasm of normal cells while in tumor cells it was localized mainly in the nucleus.(1) These differences in the localization pattern were suggested to be the main mechanism by which normal cells show resistance to Apoptin-mediated cell killing. Although the mechanism of action of Apoptin is presently unknown, it seems to function by the induction of programmed cell death (PCD) after translocation from the cytoplasm to the nucleus and arresting the cell cycle at G2/M, possibly by interfering with the cyclosome.(2) In addition, cancer specific phosphorylation of Threonine residue 108 has been suggested to be important for Apoptin's function to kill tumor cells.(3) In contrast to the large number of publications reporting that nuclear localization, induction of PCD and phosphorylation of Apoptin is restricted to cancer cells, several recent studies have shown that Apoptin has the ability to migrate to the nucleus and induce PCD in some of the normal cell lines tested. There is evidence that high protein expression levels as well as the cellular growth rate may influence Apoptin's ability to specifically kill tumor cells. Thus far both in vitro and in vivo studies indicate that Apoptin is a powerful apoptosis inducing protein with a promising prospective utility in cancer therapy. However, here we show that several recent findings contradict some of the earlier results on the tumor specificity of Apoptin, thus creating some controversy in the field. The aim of this article is to review the available data, some published and some unpublished, which either agree or contradict the reported "black and white" tumor cell specificity of Apoptin. Understanding what factors appear to influence its function should help to develop Apoptin into a potent anti-cancer agent.

Initial signaling of the fibronectin receptor (alpha5beta1 integrin) in hepatic stellate cells is independent of tyrosine phosphorylation.

BACKGROUND/AIMS: Activation of hepatic stellate cells (HSC) plays an integral role in hepatic fibrosis. HSC activation increases fibronectin (alpha(5)beta(1)) receptor expression and interactions between alpha(5)beta(1) and the extracellular matrix increase collagen synthesis. It is unclear how signaling by the alpha(5)beta(1) receptor initiates these changes. We aimed to determine the signaling cascade after alpha(5)beta(1) stimulation in activated HSC. METHODS: HSC were isolated from male Sprague-Dawley rats. Activated HSC were exposed to beads coated with fibronectin (ligand for alpha(5)beta(1)) or D-polylysine (inert control). HSC were stained with FTC-labeled antibodies against classes of signaling molecules. Tyrosine phosphorylation was blocked using genistein or herbimycin A. The fraction of beads with localized immunostaining (indicating accumulation of signaling protein) was determined. RESULTS: The majority of cytoskeletal proteins, Src substrates, Src kinases and members of the ERK and JNK signaling molecule families require actin cytoskeletal organization and tyrosine-kinase-mediated phosphorylation to accumulate. Several proteins (e.g. tensin, FAK) accumulated in the absence of tyrosine phosphorylation. CONCLUSIONS: The alpha(5)beta(1) integrin-ligand interaction induces accumulation of cytoskeletal molecules, activating multiple kinase pathways. Initial integrin signaling by alpha(5)beta(1) are associated with cytoskeletal proteins and are independent of tyrosine phosphorylation. We suggest that there may be cytoskeletal changes that may be targeted to diminish HSC activation.

Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays.

The Paramyxovirus respiratory syncytial virus (RSV) is the primary etiologic agent of serious epidemic lower respiratory tract disease in infants, immunosuppressed patients, and the elderly. Lower tract infection with RSV is characterized by a pronounced peribronchial mononuclear infiltrate, with eosinophilic and basophilic degranulation. Because RSV replication is restricted to airway epithelial cells, where RSV replication induces potent expression of chemokines, the epithelium is postulated to be a primary initiator of pulmonary inflammation in RSV infection. The spectrum of RSV-induced chemokines expressed by alveolar epithelial cells has not been fully investigated. In this report, we profile the kinetics and patterns of chemokine expression in RSV-infected lower airway epithelial cells (A549 and SAE). In A549 cells, membrane-based cDNA macroarrays and high-density oligonucleotide probe-based microarrays identified inducible expression of CC (I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, and MIP-1 alpha and -1 beta), CXC (GRO-alpha, -beta, and -gamma, ENA-78, interleukin-8 [IL-8], and I-TAC), and CX(3)C (Fractalkine) chemokines. Chemokines not previously known to be expressed by RSV-infected cells were independently confirmed by multiprobe RNase protection assay, Northern blotting, and reverse transcription-PCR. High-density microarrays performed on SAE cells confirmed a similar pattern of RSV-inducible expression of CC chemokines (Exodus-1, RANTES, and MIP-1 alpha and -1 beta), CXC chemokines (I-TAC, GRO-alpha, -beta, and -gamma, and IL-8), and Fractalkine. In contrast, TARC, MCP-1, and MDC were not induced, suggesting the existence of distinct genetic responses for different types of airway-derived epithelial cells. Hierarchical clustering by agglomerative nesting and principal-component analyses were performed on A549-expressed chemokines; these analyses indicated that RSV-inducible chemokines are ordered into three related expression groups. These data profile the temporal changes in expression by RSV-infected lower airway epithelial cells of chemokines, chemotactic proteins which may be responsible for the complex cellular infiltrate in virus-induced respiratory inflammation.

Potential double-flipping mechanism by E. coli MutY.

To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.

The peroxisomal proliferator clofibrate enhances the hepatic cytoplasmic movement of fatty acids in rats.

The role of cytosolic fatty acid binding protein (FABP) in cellular fatty acid metabolism remains poorly defined. The intracellular movement of fatty acids is thought to be facilitated through codiffusion with FABP. Peroxisomal proliferators like clofibrate induce FABP and may stimulate fatty acid use by increasing cytoplasmic diffusion rates. Our aim was to determine if induction of FABP by clofibrate increases the cytoplasmic transport of a fluorescent fatty acid NBD-stearate. Male rats were fed clofibrate for 9 days to increase the hepatic concentration of FABP. Hepatocytes were isolated using collagenase perfusion. Cytosolic FABP was measured by enzyme-linked immunosorbent assay (ELISA) using a specific antibody to hepatic FABP. Two-dimensional laser photobleaching (FRAP) was used to measure the cytoplasmic movement of NBD-stearate in hepatocytes. Cytoplasmic transport of NBD-stearate occurred by isotropic diffusion with no evidence for convective or directed transport. Treatment with clofibrate increased FABP levels, the fraction of NBD-stearate found in cytosol, and the observed cytoplasmic diffusion rate. The increase in cytoplasmic movement exceeded that predicted from FABP binding changes suggesting that clofibrate also enhances fatty acid diffusion within intracellular membranes. Nonspecific effects of clofibrate on cytoplasmic viscosity or pore size were not observed. These data suggest that clofibrate treatment induces cytosolic FABP and stimulates the intracellular movement of fatty acids by 2 distinct mechanisms. Soluble proteins like FABP promote the cytoplasmic transport of fatty acids by increasing diffusion. These data further support a role for intracellular binding proteins in facilitating the intracellular movements of fatty acids.

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex.

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate.

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.

Structural similarities between MutT and the C-terminal domain of MutY.

One of the functions of MutY from Escherchia coli is removal of adenine mispaired with 7,8-dihydro-8-oxoguanine (8-oxoG), a common lesion in oxidatively damaged DNA. MutY is composed of two domains: the larger N-terminal domain (p26) contains the catalytic properties of the enzyme while the C-terminal domain (p13) affects substrate recognition and enzyme turnover. On the basis of sequence analyses, it has been recently suggested that the C-terminal domain is distantly related to MutT, a dNTPase which hydrolyzes 8-oxo-dGTP [Noll et al. (1999) Biochemistry 38, 6374-6379]. We have studied the solution structure of the C-terminal domain of MutY by NMR and find striking similarity with the reported solution structure of MutT. Despite low sequence identity between the two proteins, they have similar secondary structure and topology. The C-terminal domain of MutY is composed of two alpha-helices and five beta-strands. The NOESY data indicate that the protein has two beta-sheets. MutT is also a mixed alpha/beta protein with two helices and two beta-sheets composed of five strands. The secondary structure elements are similarly arranged in the two proteins.

How Do Proteins Recognize DNA? Solution Structure and Local Conformational Dynamics of Lac Operators by 2D NMR.

Abstract The NMR structures of the symmetrical lac operator DNA fragment, d(TGTGAGCGCTCACA)(2) and it's mutant, d(TATGAGCGCTCATA)(2), were determined by the MORASS hybrid relaxation matrix/restrained molecular dynamics methodology. The (1)H chemical shifts of nearly all of the non-exchangeable protons were assigned using standard two-dimensional NMR techniques. Ultimately, 181 NOE volumes/strand were used in the final MORASS structural determination for each molecule. Both model built A- and B-form DNA starting geometries were used which converged to final structures giving 1.85Å and 1.32Å RMSD for the wild-type and mutant operators respectively. An excellent agreement between experimental NOESY data with that calculated from the final structures was achieved. The sequence dependence of the DNA backbone torsional angle conformational dynamics was further examined using trajectories from four 500 ps AMBER PMES molecular dynamics calculations performed on the final NMR structures. These are discussed as well as the experimental vs. calculated JH3'-P coupling constants and their relation to backbone dynamics.

Aptamers containing thymidine 3'-O-phosphorodithioates: synthesis and binding to nuclear factor-kappaB.

Aptamers targeting NF-kappaB containing thymidine 3'-O-phosphorodithioates in selected positions of an oligonucleotide duplex were synthesized. Binding affinities to NF-kappaB varied with the number and positions of the dithioate backbone substitutions. One of the aptamers showed specific binding to a single NF-kappaB dimer in cell culture extracts.

Hybrid-hybrid matrix structural refinement of a DNA three-way junction from 3D NOESY-NOESY.

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (approximately 60 degrees) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (+/- 20 degrees) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method.

Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP.

Nefazodone-induced liver failure: report of three cases.

BACKGROUND: Liver failure is a rare but devastating result of drug toxicity. OBJECTIVE: To describe three cases of subfulminant liver failure that were probably caused by nefazodone, a new antidepressant that is a synthetically derived phenylpiperazine. DESIGN: Case series. SETTING: Two university medical centers and a children's hospital. PATIENTS: Three women 16 to 57 years of age. INTERVENTION: Two patients underwent liver transplantation; the third was listed for transplantation but subsequently improved. MEASUREMENT: Liver biopsy. RESULTS: Nefazodone was administered for 14 to 28 weeks before the onset of symptoms. The duration of jaundice before onset of encephalopathy ranged from 4 to 6 weeks. All cases of liver failure had similar histologic appearance, with prominent necrosis in the centrolobular areas (zone 3). One patient had successful liver transplantation, one underwent transplantation but died, and one improved without transplantation. The temporal onset of disease after the start of nefazodone therapy suggested severe hepatocellular injury caused by the drug. CONCLUSIONS: Because nefazodone seems to cause severe hepatocellular injury in an idiosyncratic manner, routine liver chemistries should be performed before starting nefazodone therapy and patients should be monitored regularly. Therapy should be discontinued if liver enzyme concentrations become abnormal.

Hepatic iron concentration in hereditary hemochromatosis does not saturate or accurately predict phlebotomy requirements.

OBJECTIVE: Biochemical measurement of the hepatic iron concentration (HIC) is essential for the diagnosis of hereditary hemochromatosis (HH). The aim of this study was to determine whether the HIC at the time of diagnosis could predict the subsequent phlebotomy requirements and to determine whether saturation of HIC occurred in HH. METHODS: Fifty-four patients (32 male, 22 female) with homozygous HH were evaluated, and HIC was measured in liver biopsies. Patients were subjected to weekly phlebotomy (500 ml) until the transferrin saturation was <50% and/or the serum ferritin concentration was <50 microg/L. The relationship between HIC and total body iron stores (as measured by phlebotomy requirements) was determined using both linear and nonlinear (sigmoidal model) least squares regression. RESULTS: The HIC ranged from 3,742 to 41,040 microg/g dry wt. A linear relationship between HIC and total body iron stores (iron removed, IR, g) best described the data both in male (HIC = 1986 IR - 3494; r = 0.83; p < 0.001) and female HH patients (HIC = 1251 IR + 2690; r = 0.75; p < 0.001). Men required eight more phlebotomies (2 g iron) on average, compared with women, to reach normal iron stores. There was no evidence of saturation of hepatic iron levels at higher total body iron stores. However, accurate prediction of individual phlebotomy requirements based on the HIC or serum ferritin concentration at the time of diagnosis was not possible. CONCLUSION: The phlebotomy requirement for treatment of HH cannot be accurately predicted from the initial HIC or serum ferritin level. Within the range examined, hepatic iron deposition did not saturate in HH.

Sex differences in multiple steps in hepatic transport of palmitate support a balanced uptake mechanism.

Hepatic clearance of long-chain fatty acids is substantially faster in females than in males, a fact that may underlie known gender-related differences in lipoprotein metabolism and associated disease states. To further investigate the transport steps responsible for this difference, we used a novel method combining multiple-indicator dilution and steady-state measurements of palmitate extraction from albumin solutions. We found that cytoplasmic transport of palmitate is sufficiently slow (diffusion constants 9.0 and 5.9 x 10(-9) cm2/s for male and female liver, respectively) that the steady-state concentration of palmitate in the center of the cell should be approximately 0.5 of that found in the cytoplasm just beneath the plasma membrane. Previous studies in cultured liver cells using nonphysiological fatty acids have shown more rapid cytoplasmic transport in females. This sex difference reflects higher concentrations of cytosolic fatty acid-binding protein, which acts as a carrier system to transport fatty acids across cell water layers. The current study confirmed slow cytoplasmic diffusion rates in intact perfused rat liver using a physiological fatty acid and found a similar female-to-male ratio. Female liver also had a greater influx rate constant and a larger vascular volume than male liver but had a similar rate of metabolism. Rapid cytoplasmic diffusion enhances movement of palmitate into deeper layers of the cell cytoplasm, thus reducing efflux. The larger sinusoidal volume in females not only permits more dissociation of palmitate from albumin within the sinusoids but also may generate a greater permeability-surface area product. These multiple sex-related differences combine to produce a nearly twofold greater steady-state uptake rate by female liver.

Liver transplantation. Who should be referred--and when?

Orthotopic liver transplantation is a highly effective treatment for patients with end-stage cirrhosis. At most centers, 5-year survival rates are about 85%. However, donor organs continue to be in short supply, and the issue of patient selection has become increasingly important. The primary care physician plays an important role in referring the patient early--before irreversible complications arise.