Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.     

Metabolomics of the Tumor Microenvironment in Pediatric Acute Lymphoblastic Leukemia

The tumor microenvironment is emerging as an important therapeutic target. Most studies, however, are focused on the protein components, and relatively little is known of how the microenvironmental metabolome might influence tumor survival. In this study, we examined the metabolic profiles of paired bone marrow (BM) and peripheral blood (PB) samples from 10 children with acute lymphoblastic leukemia (ALL). BM and PB samples from the same patient were collected at the time of diagnosis and after 29 days of induction therapy, at which point all patients were in remission. We employed two analytical platforms, high-resolution magnetic resonance spectroscopy and gas chromatography-mass spectrometry, to identify and quantify 102 metabolites in the BM and PB. Standard ALL therapy, which includes l-asparaginase, completely removed circulating asparagine, but not glutamine. Statistical analyses of metabolite correlations and network reconstructions showed that the untreated BM microenvironment was characterized by a significant network-level signature: a cluster of highly correlated lipids and metabolites involved in lipid metabolism (p<0.006). In contrast, the strongest correlations in the BM upon remission were observed among amino acid metabolites and derivatives (p<9.2×10^-10). This study provides evidence that metabolic characterization of the cancer niche could generate new hypotheses for the development of cancer therapies.     

Morphological and genetic analysis of three new species of Ceratomyxa Thélohan, 1892 (Myxozoa: Myxosporea) from carcharhinid sharks off Australia

Three new species of Ceratomyxa Thélohan, 1892 are described from the gall-bladders of two species of carcharhinid sharks collected off Heron and Lizard Islands on the Great Barrier Reef, Australia. Ceratomyxa carcharhini n. sp. and C. melanopteri n. sp. are described from Carcharhinus melanopterus (Quoy & Gaimard), and Ceratomyxa negaprioni n. sp. is described from Negaprion acutidens (Rüppell). These species are the first ceratomyxids reported from Australian elasmobranchs, and this is the first paper to formally characterise a novel Ceratomyxa species from an elasmobranch using both morphology and small subunit ribosomal DNA sequence data. Maximum parsimony and Bayesian inference analyses of the SSU rDNA dataset revealed that ceratomyxids from elasmobranchs form a sister clade to that of species infecting marine teleosts and Palliatus indecorus Schulman, Kovaleva & Dubina, 1979. Furthermore, the only sequenced freshwater ceratomyxid, Ceratomyxa shasta Noble, 1950, fell outside the overall marine ceratomyxid clade. These data show that Ceratomyxa, as currently recognised, is polyphyletic and ignites discussion on whether Ceratomyxa should be split. However, further taxon sampling, particularly in freshwater systems, is required to establish relevant biological divisions within the genus.     

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.     

Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor

We demonstrated that ginsenoside-Re (Re), a pharmacological active component of ginseng, is a functional ligand of glucocorticoid receptor (GR) using competitive ligand-binding assay (IC₅₀ = 156.6 nM; K_d = 49.7 nM) and reporter gene assay. Treatment with Re (1 μM) raises intracellular Ca²⁺ ([Ca²⁺]_i) and nitric oxide (NO) levels in human umbilical vein endothelial cells as measured using fura-2 and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, respectively. Western blot analysis shows that Re increased phosphorylation of endothelial nitric oxide synthase. These effects were abolished by GR antagonist RU486, siRNA targeting GR, non-selective cation channel blocker 2-aminoethyldiphenylborate, or in the absence of extracellular Ca²⁺, indicating Re is indeed an agonistic ligand for the GR and the activated GR induces rapid Ca²⁺ influx and NO production in endothelial cells.     

Targeting base excision repair to improve cancer therapies

Most commonly used cancer therapies, particularly ionizing radiation and certain classes of cytotoxic chemotherapies, cause cell death by damaging DNA. Base excision repair (BER) is the major system responsible for the removal of corrupt DNA bases and repair of DNA single strand breaks generated spontaneously and induced by exogenous DNA damaging factors such as certain cancer therapies. In this review, the physico-chemical properties of the proteins involved in BER are discussed with particular emphasis on molecular mechanisms coordinating repair processes. The aim of this review is to apply extensive knowledge that currently exists regarding the biochemical mechanisms involved in human BER to the molecular biology of current therapies for cancer. It is anticipated that the application of this knowledge will translate into the development of novel effective therapies for improving existing treatments such as radiation therapy and oxaliplatin chemotherapy.     

Increased spontaneous tone in renal arteries of spontaneously hypertensive rats

The spontaneous tone of vascular smooth muscle is augmented in hypertension. The present study examined the role of nitric oxide (NO), cyclooxygenase (COX), thromboxane A(2)/prostanoid (TP) and PGE(2)/prostanoid (EP-1) receptors, reactive oxygen species, and large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the regulation of spontaneous tone in renal arteries of young and mature Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Rings of arteries, with and without endothelium, were suspended in a myograph for isometric force recording. Spontaneous tone (increase above initial tension) was observed only in arteries of mature SHR and was greater in arteries without endothelium. N(omega)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthases) induced larger contractions in arteries of SHR than WKY. Indomethacin (a COX inhibitor), SC-19220 (an EP-1 receptor antagonist), and terutroban (a TP receptor antagonist) reduced the L-NAME-evoked contractions. Tiron (a superoxide anion scavenger), catalase (an enzyme that degrades H(2)O(2)), and deferoxamine (a hydroxyl radical scavenger) augmented the L-NAME-induced contractions in arteries of mature SHR. Charybdotoxin (a BK(Ca) channel blocker) caused contractions in arteries of mature SHR without endothelium and in arteries with endothelium incubated with L-NAME. A decreased protein level of endothelial NO synthase, an increased release of prostacyclin, and an increased expression of EP-1 receptors were observed in arteries of mature SHR. The present study suggests that spontaneous tone is precipitated by age and hypertension. The reduced production of NO, leading to decreased activation of BK(Ca) channels, may leave the actions of endogenous vasoconstrictors unopposed. COX products that activate EP-1 and TP receptors are involved in the development of spontaneous tone.