A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines. Author Keywords: Helminths; Trematodes; Parasites; Cathepsins; Proteases; Vaccines; Immunology; Biochemistry

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4(-/-) mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4(-/-) c-Kit(+) fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4(-/-) mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4(-/-) and Hoxb1-b9(-/-) fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-KIt(+) Hoxb1-bg(-/-) fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4, -a11, and -c4. Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent.

Expression of osteopontin coregulators in primary colorectal cancer and associated liver metastases

Background: A transcription regulatory complex (TRC) that includes Ets1, Ets2, PEA3 and ß-catenin/T-cell factors regulates osteopontin (OPN) that is implicated in colorectal cancer (CRC) dissemination. The consistency of OPN transcriptional control between primary CRC and metastases is unclear. This study investigates expression and prognostic significance of the OPN–TRC in primary human CRC and associated colorectal liver metastases (CRLM).

Methods: Osteopontin–TRC factors were assayed by digital microscopy in 38 primary CRCs and matched CRLM specimens and assessed against clinical prognosis.

Results: In primary CRC, OPN expression intensity correlated with that of its co-activators, PEA3 (r=0.600; P<0.01), Ets1 (r=0.552; P<0.01), Ets2 (r=0.521; P<0.01) and had prognostic significance. Osteopontin intensity in primary CRC inversely correlated with the interval between diagnosis and resection of CRLM. Overall OPN intensity was lower in CRLM than primary CRC and correlations with co-activators were weaker, for example, Ets1 (P=0.047), PEA3 (P=0.022) or nonsignificant (Ets2). The ratio of OPN expression in CRLM vs primary CRC had prognostic significance.

Conclusion: This study supports transcriptional control of OPN by known coregulators in both primary and secondary CRC. Weaker associations in CRLM suggest involvement of other unknown factors possibly from the liver microenvironment or resulting from additional genetic or epigenetic changes that drive tumour metastatic capability in OPN transcriptional control.

C/EBP alpha Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation

BACKGROUND & AIMS: C/EBP alpha (cebpa) is a putative tumor suppressor. However, initial results indicated that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs). The regulation and function of C/EBP alpha was investigated in HCC cell lines to clarify its role in liver carcinogenesis. METHODS: The regulation of C/EBP alpha expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBP alpha expression was knocked-down by small interfering RNA or short hairpin RNA. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays. RESULTS: Cebpa was up-regulated at least 2-fold in a subset (approximately 55%) of human HCCs compared with adjacent non tumor tissues. None of the up-regulated samples were positive for hepatitis C infection. The HCC cell lines Hep3B and Huh7 expressed high, PLC/PRF/5 intermediate, HepG2 and HCC-M low levels of C/EBP alpha, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBP alpha gene in HCCs and cell lines. C/EBP alpha was localized to the nucleus and functional in Hep3B and Huh7 cells; knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. Epigenetic mechanisms including DNA methylation, and the binding of acetylated histone H3 to the CEBP alpha promoter-regulated cebpa expression in the HCC cells. CONCLUSIONS: C/EBP alpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBP alpha may be amenable to epigenetic regulation to improve treatment outcomes.

Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6) M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

ULTRASTRUCTURAL-LOCALIZATION OF FMRFAMIDE-IMMUNOREACTIVITY AND PANCREATIC POLYPEPTIDE-IMMUNOREACTIVITY WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE LIVER FLUKE, FASCIOLA-HEPATICA (TREMATODA, DIGENEA)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMRFamide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode, Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and 'giant' nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.

LOCALIZATION OF ACTIN IN THE LIVER FLUKE, FASCIOLA-HEPATICA

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1 % (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

A plasma membrane Ca2+-ATPase (PMCA) from the liver fluke, Fasciola hepatica

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an a-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Misonidazole binding in murine liver tissue: a marker for cellular hypoxia in vivo

The hepatic microcirculation is believed to cause variable cellular oxygenation within the organ. In this study a marker of cellular hypoxia was used to demonstrate liver oxygen tension gradients in vivo. Covalent binding of misonidazole adducts to cellular macromolecules is enhanced by hypoxia. Autoradiographs of liver from mice treated with radiolabeled misonidazole demonstrated enhanced binding of adducts within hepatocytes surrounding hepatic veins. Livers from both hypoxic and normal mice had characteristic autoradiographic grain patterns reflecting regional oxygen tension variation in vivo. Differential binding of misonidazole adducts formed in hypoxic cells could have an application in studies of liver physiology and biochemistry.

Localisation of actin in the liver fluke, Fasciola hepatica

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD+/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD+/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD+/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD+/NADH redox potential may be of potential therapeutic benefit in the clinical setting.