In situ zymography: topographical considerations

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic Variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity. (C) 2001 Elsevier Science B.V. All rights reserved.

Telehealth and the diagnosis and management of cardiac disease

The financial and personal burden of chronic cardiac disease is high. Costs are likely to increase over the next few decades. Promising applications of telehealth have appeared in the diagnosis and management of cardiac disease and there are indications that telehealth services can improve the management of chronic cardiac disease as well as extend services to remote and rural populations. Telehealth has been applied to the capture of symptoms of cardiac disease with electrocardiography and echocardiography, to the management and rehabilitation of recently discharged patients, and in peer-to-peer consultation where remote expertise can facilitate diagnosis. Telehealth promises cost reductions in service delivery, although there is a need for properly controlled cost-effectiveness trials to underpin telehealth with a firm evidence base.

Premature calvarial synostosis and epidermal hyperplasia (Beare-Stevenson syndrome-like anomalies) resulting from a P250R missense mutation in the gene encoding fibroblast growth factor receptor 3

We report on a patient with a severe premature calvarial synostosis and epidermal hyperplasia. The phenotype was consistent with that of a mild presentation of Beare-Stevenson syndrome but molecular analysis of the IgIII-transmembrane linker region and the transmembrane domain of the gene encoding the FGFR2 receptor, revealed wild-type sequence only. Subsequently, molecular analysis of the FGFR3 receptor gene identified a heterozygous P250R missense mutation in both the proposita and her mildly affected father. This communication extends the clinical spectrum of the FGFR3 P250R mutation to encompass epidermal hyperplasia and documents the phenomenon of activated FGFR receptors stimulating common downstream developmental pathways, resulting in overlapping clinical outcomes. (C) 2001 Wiley-Liss, Inc.

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-I and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.

Quantitative evaluation of altered hepatic spaces and membrane transport in fibrotic rat liver

Four animal models were used to quantitatively evaluate hepatic alterations in this study: (1) a carbon tetrachloride control group (phenobarbital treatment only), (2) a CCl4-treated group (phenobarbital with CCl4 treatment), (3) an alcohol-treated group (liquid diet with alcohol treatment), and (4) a pair-fed alcohol control group (liquid diet only). At the end of induction, single-pass perfused livers were used to conduct multiple indicator dilution (MID) studies. Hepatic spaces (vascular space, extravascular albumin space, extravascular sucrose space, and cellular distribution volume) and water hepatocyte permeability/surface area product were estimated from nonlinear regression of outflow concentration versus time profile data. The hepatic extraction ratio of H-3-taurocholate was determined by the nonparametric moments method. Livers were then dissected for histopathologic analyses (e.g., fibrosis index, number of fenestrae). In these 4 models, CCl4-treated rats were found to have the smallest vascular space, extravascular albumin space, H-3-taurocholate extraction, and water hepatocyte permeability/surface area product but the largest extravascular sucrose space and cellular distribution volume. In addition, a linear relationship was found to exist between histopathologic analyses (fibrosis index or number of fenestrae) and hepatic spaces. The hepatic extraction ratio of H-3-taurocholate and water hepatocyte permeability/surface area product also correlated to the severity of fibrosis as defined by the fibrosis index. In conclusion, the multiple indicator dilution data obtained from the in situ perfused rat liver can be directly related to histopathologic analyses.

The Mallory body as an aggresome: In vitro studies

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+ 1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+ 1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK 18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+ 1. (C) 2002 Elsevier Science.

Enterohepatic circulation - Physiological, pharmacokinetic and clinical implications

Enterohepatic recycling occurs by biliary excretion and intestinal reabsorption of a solute, sometimes with hepatic conjugation and intestinal deconjugation. Cycling is often associated with multiple peaks and a longer apparent half-life in a plasma concentration-time profile. Factors affecting biliary excretion include drug characteristics (chemical structure, polarity and molecular size), transport across sinusoidal plasma membrane and canniculae membranes, biotransformation and possible reabsorption from intrahepatic bile ductules. Intestinal reabsorption to complete the enterohepatic cycle may depend on hydrolysis of a drug conjugate by gut bacteria. Bioavailability is also affected by the extent of intestinal absorption, gut-wall P-glycoprotein efflux and gut-wall metabolism. Recently, there has been a considerable increase in our understanding of the role of transporters, of gene expression of intestinal and hepatic enzymes, and of hepatic zonation. Drugs, disease and genetics may result in induced or inhibited activity of transporters and metabolising enzymes. Reduced expression of one transporter, for example hepatic canalicular multidrug resistance-associated protein (MRP) 2, is often associated with enhanced expression of others, for example the usually quiescent basolateral efflux MRP3, to limit hepatic toxicity. In addition, physiologically relevant pharmacokinetic models, which describe enterohepatic recirculation in terms of its determinants (such as sporadic gall bladder emptying), have been developed. In general, enterohepatic recirculation may prolong the pharmacological effect of certain drugs and drug metabolites. Of particular importance is the potential amplifying effect of enterohepatic variability in defining differences in the bioavailability, apparent volume of distribution and clearance of a given compound. Genetic abnormalities, disease states, orally administered adsorbents and certain coadministered drugs all affect enterohepatic recycling.

A compartmental model of hepatic disposition kinetics: 1. Model development and application to linear kinetics

The conventional convection-dispersion model is widely used to interrelate hepatic availability (F) and clearance (Cl) with the morphology and physiology of the liver and to predict effects such as changes in liver blood flow on F and Cl. The extension of this model to include nonlinear kinetics and zonal heterogeneity of the liver is not straightforward and requires numerical solution of partial differential equation, which is not available in standard nonlinear regression analysis software. In this paper, we describe an alternative compartmental model representation of hepatic disposition (including elimination). The model allows the use of standard software for data analysis and accurately describes the outflow concentration-time profile for a vascular marker after bolus injection into the liver. In an evaluation of a number of different compartmental models, the most accurate model required eight vascular compartments, two of them with back mixing. In addition, the model includes two adjacent secondary vascular compartments to describe the tail section of the concentration-time profile for a reference marker. The model has the added flexibility of being easy to modify to model various enzyme distributions and nonlinear elimination. Model predictions of F, MTT, CV2, and concentration-time profile as well as parameter estimates for experimental data of an eliminated solute (palmitate) are comparable to those for the extended convection-dispersion model.