Fulvio Micali

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high‐affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of ≈ 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti‐sequence bilitranslocase antibodies, the protein‐modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.

EXPRESSION OF BILITRANSLOCASE IN THE VASCULAR ENDOTHELIUM AND ITS FUNCTION AS A FLAVONOID TRANSPORTER

AIMS Ingestion of flavonoid-rich beverages acutely affects endothelial function, causing vasodilation. This effect might be dependent on flavonoid transport into the endothelium. We investigated flavonoid uptake into vascular endothelial cells and whether this was mediated by bilitranslocase (TC 2.A.65.1.1), a bilirubin-specific membrane carrier that also transports various dietary flavonoids. METHODS AND RESULTS Human and rat aortic primary endothelial cells as well as Ea.hy 926 cells were found to express bilitranslocase, as assessed by immunocytochemistry and immunoblotting analysis using anti-sequence bilitranslocase antibodies targeting two distinct extracellular epitopes of the carrier. Bilitranslocase function was tested by measuring the rate of bromosulfophthalein (a standard bilitranslocase transport substrate) uptake into endothelial cells and was inhibited not only by bilitranslocase antibodies but also by quercetin (a flavonol). Similarly, uptake of both quercetin and malvidin 3-glucoside (an anthocyanin) were also found to be antibody-inhibited. Quercetin uptake into cells was inhibited by bilirubin, suggesting flavonoid uptake via a membrane pathway shared with bilirubin. CONCLUSION The uptake of some flavonoids into the vascular endothelium occurs via the bilirubin-specific membrane transporter bilitranslocase. This offers new insights into the vascular effects of both flavonoids and bilirubin.

The spatial distribution of calcium in alginate gel beads analysed by synchrotron-radiation induced X-ray emission (SRIXE)

Abstract The spatial distribution of calcium ions in alginate gel spheres was investigated by synchrotron radiation induced X-ray emission. X-ray emission spectra were obtained from 1 mm spherical calcium alginate beads gelled either in the presence or in the absence of sodium ions. Evidence was found for an inhomogeneous distribution of calcium ions with the highest concentration at the surface, and the lowest concentration in the core of the spheres, which reflected the spatial distribution of alginate in the gel spheres. The form and the steepness of the calcium concentration gradient was strongly affected by the presence of sodium ions.

Differential Splicing of the Phospholipid Hydroperoxide Glutathione Peroxidase Gene in Diploid and Haploid Male Germ Cells in the Rat

Abstract Phospholipid hydroperoxide glutathione peroxidase (PHGPx, 20 kDa) and sperm nuclei glutathione peroxidase (snGPx, 34 kDa) are two selenoproteins present in mammalian testis and epididymal spermatozoa. They originate from the differential splicing of the PHGPx gene and appear to play important roles in sperm physiology. To determine the stages of spermatogenesis in which they are present, we compared the expression pattern of these two enzymes in highly purified populations of germ cells during specific phases of differentiation. In Northern and Western blotting experiments, both PHGPx transcript and protein were markedly expressed in pachytene spermatocytes and round spermatids. In contrast, the testis-specific snGPx was detected at both the mRNA and protein level only in haploid round spermatids. Accordingly, the developmental analysis of testicular RNAs from rats of different ages first revealed the appearance of PHGPx and snGPx transcripts at Day 20 and Day 30, respectively. Furthermore, both meiotic and postmeiotic cells contained catalytically active PHGPx/snGPx, with higher activity in the haploid cells. The intracellular distribution of PHGPx in mitochondria and nuclei of meiotic cells was demonstrated by immunocytochemical electron microscopy and Western blotting. These findings provide evidence that the PHGPx gene is differentially spliced during the meiotic prophase and haploid cell phases of spermatogenesis.

Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin‐binding proteins was tested by searching for peptides containing the bilirubin‐binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.

Structural investigations of cross-linked hyaluronan

Structural properties of several cross-linked hyaluronan derivatives, obtained by scanning electron microscopy, monodimensional NMR microscopy and small angle X-ray scattering of synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid, used as a reference material. The experimental results, obtained in different media, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of denser polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover, the presence of polymer junction zones gave rise to the appearance of a well-defined correlation peak in the pattern of intensity of the scattered X-radiation.

Low variability of the protein species synthesized byDrosophila melanogaster embryos

SummaryStainable proteins as well as newly synthesized polypeptide chains of proteins extracted fromDrosophila melanogaster embryos were analyzed by two-dimensional gel electrophoresis. The following developmental stages were studied: unfertilized eggs, early nuclear multiplication (25 min average age), late nuclear multiplication (105 min), cellular blastoderm (165 min), gastrula (4 h), mesodermal segmentation (6 h) and muscleattachment (8 h). One hundred and fifty stainable spots were present at all developmental stages and were all also synthesized during development, with the exception of 5 unkown proteins and the three yolk proteins. Out of 400 proteins which were labelled by35S-methionine, only 5% showed a reproducible pattern of variable synthesis. Three proteins appeared upon fertilization. The early nuclear multiplication stage showed the largest number of labelled spots while the lowest number of labelled spots was observed at blastoderm formation. The pattern of synthesis of a few specific proteins was also followed. Actin I was synthesized only at 8 h, actin II and actin III were synthesized at all stages. β-tubulin was synthesized at all stages, while we observed a reduction, if not a cessation, of synthesis of α-tubulin at 105 min, 165 min and 4 h of development. Non heat-shock embryos synthesized a large amount of heat-shock protein (hsp) 84 at 25 min while hsp 70 and 68 were first detected after 4 h of development. Though it is generally accepted that the embryonic genome is activated at blastoderm formation we did not observe a parallel increase in protein species. It is possible that protein synthesis on the new transcripts is below the detection limit of the technique. Alternatively the embryonic messages may gradually substitute preexisting maternal messages or only become available for translation some time after they are transcribed.

Immunolocalisation of bilitranslocase in mucosecretory and parietal cells of the rat gastric mucosa.

SummaryBilitranslocase is a plasma membrane carrier localised at the vascular pole of the rat liver cell, where it mediates uptake of organic anions from the blood into the liver. This carrier is also present in the epithelium of the rat gastric mucosa, with similar molecular mass and functional properties. An immunohistochemical study reveals that both the mucus-secreting cells of the gastric pit and the H+-secreting parietal cells express bilitranslocase. These data point to a possible role of bilitranslocase and of its food-borne substrates (anthocyanins and nicotinic acid) in regulating the function and the permeability of the gastric mucosa.

Expression and characterization of human‐elastin‐repeat‐based temperature‐responsive protein polymers for biotechnological purposes

Rapid progress has been made in the design and synthesis of oligomers and polymers that emulate the properties of natural proteins. Molecular bioengineering offers the chance to design and produce artificial polymeric proteins with tailored polymeric properties. The elastin‐like polypeptides are a well‐defined family of polymers with noteworthy characteristic based on the VPGVG repeated motif of bovine elastin. In the human homologue, the most regular sequence is represented by the repetition of the VAPGVG hexapeptidic motif. On the basis of this sequence, a synthetic gene has been designed, cloned and expressed in Escherichia coli to obtain artificial protein polymers. The rapid one‐step in‐frame cloning of any biologically active sequence can be achieved directly in the expression vector, allowing further improvement of the potential of the resulting product.

NOVEL BIOMATERIALS BASED ON CROSS-LINKED HYALURONAN: STRUCTURAL INVESTIGATIONS

ABSTRACT Structural properties of several cross-linked hyaluronan derivatives, obtained by electron microscopy, monodimensional NMR microscopy and low angle X-ray scattering by synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid used as reference material. The experimental results, obtained in different media by the different techniques, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of more dense polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover the presence of polymer junction zones gave rise to the appearance of an additional correlation peak in the pattern of the intensity of the scattered X-radiation.