Confocal laser scanning microscopy (CLSM) study of hepatocytes cultured on collagen films and gels

Summary of paper describing the process whereby primary hepatocyte cultures form an integral part of many hybrid artificial liver designs, and the extracellular matrix environment of the cultures is an important factor for optimal expression of hepatocyte-specific phenotype. This study investigates the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen films and gels, and crosslinking the films and the gels with 1,6-diaminohexane (DAH) on the viability of hepatocytes cultured for 48 hours

Expression of glutathione-S-transferases in hepatocytes cultured on collagen gels of different composition

The use of primary hepatocyte cultures as in vitro models for studying xenobiotic metabolism and toxicity is limited by the loss of liver-specific differentiated functions with time in culture. This study investigates the effect of incorporating 20% chondroitin-6-sulphate (Ch6SO4), a glycosaminoglycan (GAG), into collagen gels (0.3% w/v), and crosslinking the gels with either 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or 1,6-diaminohexane (DAH) on the expression and total activity of glutathione-S-transferases (GSTs) in hepatocytes cultured for 7 days. GSTs are a family of isoenzymes. The main isoenzyme in the male rat liver is the alpha form (which exists as several subunits (Ya and Yc2 were measured here)). The pi form (subunit Yf) is not normally present in liver, but is expressed in conditions where the liver cells are de-differentiated

Kaempferol metabolism by rat hepatocytes cultured on different collagen substrates

Metabolic activity is unstable in primary hepatocyte cultures and is influenced by the matrix composition.The effect of incorporating 20% chondroitin-6-sulphate(Ch6SO4), a glycosaminoglycan, into collagen films and gels(0.3%w/v), and crosslinking the films and gels with 1,6-diaminohexane(DAH)on the glucuronidation of kaempferol by primary rat hepatocytes cultured for 48h and 7 days was investigated. Hepatocytes isolated from male Sprague-Dawley rats by collagenase perfusion were cultured (3x106/60mm Petri dish)in Chee's medium with 5% v/v foetal calf serum. Cells were incubated with 100mM kaempferol for 1h at 378C and the glucuronides(K1 and K2) were measured by HPLC. Cells cultured on collagen films formed only the K2 metabolite after 48h in culture. The addition of Ch6SO4 or DAH significantly increased the formation of this glucuronide. However, cells seeded on gels showed no metabolism after 48h in culture. By 7 days in culture, both K1 and K2 glucuronides were formed in cells on all the different films and gels. The formation of K1 glucuronide was significantly higher with the addition of Ch6SO4 to the films. K2 glucuronide was significantly higher in all of the crosslinked films compared to the plain films. Modification of collagen based substrates may improve cultured hepatocyte phenotype

Metabolic studies of hepatocytes cultured on collagen substrates modified to contain glycosaminoglycans

Bioartificial liver devices replace the function of the failing liver, using primary hepatocytes cultured in a bioreactor module. Most devices have been based on cartridge designs, but alternative designs using monolayers of cells in a flat plate bioreactor may be more efficacious. Collagen coating improves the maintenance of hepatocytes on polymeric membranes, and in this article the effect of contact with glycosaminoglycans (GAGs) on the function of hepatocytes was assessed. The effect of two different GAGs, chondroitin-6-sulfate and heparin, in the presence and absence of a cross-linking agent (1,6-diaminohexane [DAH]), on the activities of two major metabolic pathways in hepatocytes (cytochrome P-450-dependent monooxygenase activity, assessed by the hydroxylation of testosterone, and UDP-glucuronosyltransferase activity, assessed by the glucuronidation of kaempferol) cultured on collagen gels and films is presented. Testosterone metabolism was more extensive in cells cultured on collagen films than in cells cultured on gels. The addition of heparin and DAH to collagen gels supported the formation of 6β-hydroxy, 16α-hydroxy, and 2α-hydroxy testosterone by cells cultured for 48 h. The extent of glucuronidation of kaempferol was not different when comparing cells cultured on films or gels at the various times in culture; however, the ratio of formation of the two glucuronides formed, M1 and M2, was different. The combination of chondroitin- 6-sulfate and DAH increased glucuronidation of cells cultured for 7 days on both collagen films and gels. This approach may increase the expression of hepatocyte-specific functions in monolayers cultured on membranes in flat plate bioreactors

Non-nucleoside HIV-1 reverse transcriptase inhibitors di-halo-indolyl aryl sulfones achieve tight binding to drug-resistant mutants by targeting the enzyme-substrate complex.

Indolyl aryl sulfone (IAS) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) have been previously shown to effectively inhibit wild-type (wt) and drug-resistant human immunodeficiency virus type 1 (HIV-1) replication. IASs proved to act through different mechanisms of action, depending on the nature and position of their chemical substituents. Here we describe selected novel IAS derivatives (di-halo-IASs). Our results show that these compounds are selective for the enzyme-substrate complex. The molecular basis for this selectivity was a different dissociation rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the resistant enzymes carrying the single mutations Lys103Asn, Leu100Ile, and Tyr181Ile (K103N, L100I, and Y181I), we found that one compound (RS1914) dissociated from the mutated enzymes almost 10-fold slower than from the wild type RT. These results demonstrate that IASs are very flexible molecules, interacting dynamically with the viral RT, and that this property can be successfully exploited to design inhibitors endowed with an enhanced binding to common NNRTI-resistant mutants

Features, processing states and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function

Nucleocapsid (NC) is central to retroviral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NCs aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture