Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis.

HSP72 is an important marker for various environmental stresses and diseases, and many researchers need to detect HSP72 levels in various cells. We have therefore developed an assay to monitor intracellular heat-shock protein 72 expression on a microfluidic Lab-on-a-chip platform. We established this method to detect HSP72 intracellularly by antibody staining with DNA counterstaining. The Lab-on-a-chip technology is simple and efficient when performing flow cytometric assays. By permeabilizing the cells for the delivery of antibodies, we were able to show HSP72 expression after 30 min heat-shock at 44 degrees C and then at various post-incubation times at 37 degrees C. We compared our method to a conventional flow cytometer and an enzyme immunoassay technique.

Fatty acid metabolism in human breast cancer cells (MCF7) transfected with heart-type fatty acid binding protein.

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 +/- 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [14C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.

Variation of liver-type fatty acid binding protein content in the human hepatoma cell line HepG2 by peroxisome proliferators and antisense RNA affects the rate of fatty acid uptake.

The liver-type fatty acid binding protein (L-FABP), a member of a family of mostly cytosolic 14-15 kDa proteins known to bind fatty acids in vitro and in vivo, is discussed to play a role in fatty acid uptake. Cells of the hepatoma HepG2 cell line endogenously express this protein to approximately 0.2% of cytosolic proteins and served as a model to study the effect of L-FABP on fatty acid uptake, by manipulating L-FABP expression in two approaches. First, L-FABP content was more than doubled upon treating the cells with the potent peroxisome proliferators bezafibrate and Wy14,643 and incubation of these cells with [1-14C]oleic acid led to an increase in fatty acid uptake rate from 0.55 to 0.74 and 0.98 nmol/min per mg protein, respectively. In the second approach L-FABP expression was reduced by stable transfection with antisense L-FABP mRNA yielding seven clones with L-FABP contents ranging from 0.03% to 0.14% of cytosolic proteins. This reduction to one sixth of normal L-FABP content reduced the rate of [1-14C]oleic acid uptake from 0.55 to 0. 19 nmol/min per mg protein, i.e., by 66%. The analysis of peroxisome proliferator-treated cells and L-FABP mRNA antisense clones revealed a direct correlation between L-FABP content and fatty acid uptake.

Heart-type fatty acid binding protein - involvement in growth inhibition and differentiation.

Fatty acid binding proteins (FABPs) comprise a well-established family of cytoplasmic hydrophobic ligand binding proteins and are thought to be involved in lipid metabolism by binding and intracellular transport of long-chain fatty acids. However, from other studies role for FABPs in cell signalling, growth inhibition and differentiation has also been implied. In particular, the heart-type (H-FABP) is abundantly expressed in differentiated mammary gland and its relationship with a very homologous (95%) mammary derived growth inhibitor (MDGI) was disputed. Here we give a survey on the experimental evidence for the existence of such protein with growth inhibitory function. After cloning of the bovine adipocyte-type (A-)FABP cDNA from mammary gland we conclude that the reported MDGI sequence actually represents a mixture of bovine H- and A-FABP and that the MDGI function is exerted by H-FABP. We also monitored the H-FABP level during differentiation of C2C12 muscle cells from myoblasts to multiply nucleated myotubes. H-FABP expression is clearly detected after that of the transcription factor myogenin which is upregulated immediately upon onset of differentiation and after that of the typical muscle enzyme creatine kinase. This argues against an active role of H-FABP in muscle development unlike the situation in the mammary gland.

Differentiation-dependent expression of heart type fatty acid-binding protein in C2C12 muscle cells.

The aim of the present work was to establish a cell culture model for the investigation of the influence of heart type fatty acid-binding protein (H-FABP) on differentiation and lipid metabolism. Up to now no data have been reported on H-FABP in cell lines of skeletal muscle, one of the major sources of this protein in vivo. For this purpose mouse C2C12 cells were chosen, because these cells can be stimulated to differentiate in vitro from myoblasts to spontaneously contracting, multiply nucleated myotubes expressing muscle-specific proteins like creatine kinase. Analysis of the cellular proteins by two-dimensional gel electrophoresis and ELISA demonstrated that the expression of H-FABP is differentiation dependent as well in these cells. Furthermore, immunofluorescent labeling with H-FABP-specific antibodies revealed that induction of this protein occurred mainly in myotubes. Myoblasts contained only 7.1 +/- 3.1 ng H-FABP/mg soluble protein, however, upon differentiation, this value increased about 60-fold to 420 +/- 90 ng/mg (n = 4) in a mixture of myoblasts and myotubes. H-FABP from C2C12 cells was subsequently cloned and shown to be identical to the known mouse H-FABP. The induction of H-FABP during differentiation was also detected at mRNA level by probing with H-FABP-cDNA. Insulin, a known stimulator of in vitro muscle cell differentiation, led to an increased differentiation as referenced by creatine kinase activity, which is paralleled by an increased H-FABP expression. The enhancement of H-FABP expression by insulin was found to be time- and dose-dependent. The increasing H-FABP content may relate to an increasing fatty acid oxidation that has been reported for differentiated L6 cells, a related muscle cell line from rat. Such a correlation would favor a role of H-FABP in lipid metabolism.

Treatment of cystinosis with cysteamine. A pilot study determining dose and form of application.

A pilot study with cysteamine treatment was performed in three children with the nephropathic form of cystinosis. Two children underwent renal transplantation shortly before treatment. The aim of the study was to find a practicable form of application and a corresponding effective dose. Cysteamine in gelatine capsules together with 0.2% silicic acid as a dessicator turned out to be the most acceptable galenic form, compared to sirup or suppositories. Among three dosage regimens, the dosage of 50 mg/kg/day is effective as judged by the leucocyte cystine content, even if given in only three doses per day. No side effects of the cysteamine treatment (even at a dose of 90 mg/kg/day) were noted. Whether this treatment is preventing progression of disease will have to be examined either in transplanted patients by measuring non-renal parameters or in very young infants with cystinosis whose kidneys are not damaged yet.