Two novel POLG mutations causing hepatic mitochondrial DNA depletion with recurrent hypoketotic hypoglycaemia and fatal liver dysfunction.

BACKGROUND: Inherited mtDNA depletion syndromes (MDS) are a group of severe mitochondrial disorders resulting from defects in nucleus-encoded factors and often associated with severe or fatal liver failure. PATIENT: In this article, we describe the case of an 18-month-old patient with recurrent hypoketotic hypoglycaemia and fatal hepatic dysfunction with liver mtDNA depletion. METHODS: The assessment of mtDNA copy number was performed on leucocytes, liver and muscle biopsy by Quantitative Real Time PCR and total RNA from liver biopsy was used as a template to amplify the cDNA of the POLG1 gene. RESULTS: Sequence analysis identified two previously undescribed mutations (1868T>G and 2263A>G) located in the gene coding the catalytic subunit of mitochondrial DNA polymerase gamma (POLG), predicting an L623W and K755E amino acid change, respectively. Both mutations were located in the highly conserved linker region of the protein and were absent in more than 200 healthy unrelated control subjects. The identification of these two mutations allowed us to perform genetic counselling and prenatal diagnosis. CONCLUSION: Our data further expand the spectrum of POLG1 gene mutations and the unique phenotype reported (late onset isolated liver disease without lactic acidosis) increase the variability of clinical presentations associated with mutations in this gene.

Lymphatic endothelial tumors induced by intraperitoneal injection of incomplete Freund's adjuvant.

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice, only tumors of the blood vessel endothelium (haemangiomas) have been thus far reported. Here we describe a highly reproducible method for the induction of benign tumors of the lymphatic endothelial cells (lymphangiomas) in mice by intraperitoneal injection of incomplete Freund's adjuvant. Morphological and histopathological studies of the lesions revealed the presence of cells at various levels of vascular development. The lymphangiomas developed in the peritoneal cavity and expressed the endothelial markers CD31/PECAM (platelet endothelial cell adhesion molecule), CD54/ICAM-1 (InterCellular Adhesion Molecule-1), and CD102/ICAM-2, as well as the vascular endothelial growth factor (VEGF) receptor Flk-1, the endothelial cell specific receptors Tie-1 and Tie-2 and the lymphatic endothelial cell specific Flt4 receptor as shown by in situ hybridization. The Flk-1 and Flt4 receptors were also identified in immunoblots of the tumors and in cells cultured from them. When induced in beta-galactosidase knock-in Flt4(+/-) mice, the tumor endothelia could be stained blue in a number of tumor cells although the staining was of lower intensity than in normal lymphatic vessels. The tumor-derived cells could be propagated in vitro and they spontaneously differentiated, forming vessel-like structures. Murine lymphangiomas thus represent a highly reproducible and convenient source of lymphatic endothelial cells.

Hepatic glutathione determination after ethanol administration in rat: evidence of the first-pass metabolism of ethanol.

As a fraction of ingested ethanol is metabolized by gastric mucosa, different amounts of alcohol should reach the liver when the same dose is administered by oral or intravenous route. Therefore, we investigated the time-course of hepatic reduced glutathione (GSH) concentrations after intra-peritoneal or intra-gastric load of the same amount of ethanol in the rat. The test was also performed in fasted and Cimetidine-treated rats. The oral ethanol administration was followed by a less pronounced decrease and by a quicker recovery of hepatic content of GSH than after intraperitoneal route. In the fasted rat, basal hepatic GSH significantly decreased; after alcohol administration the decrease of hepatic GSH was more severe and prolonged than in the fed rat. Cimetidine was shown to be a potent inhibitor of gastric ADH. Pre-treatment with Cimetidine did not change the basal levels of hepatic GSH, but after oral ethanol load, the decrease of the hepatic GSH content was significantly (p < 0.005) more pronounced than in controls. This study demonstrates the beneficial effects of gastric ethanol metabolism on the liver. The reduced gastric ethanol metabolism, induced by fasting or by Cimetidine resulted in a decreased content and delayed recovery of liver GSH content.

Immunoreactivity of chimeric proteins carrying the HIV-1 epitope IGPGRAF. Correlation between predicted conformation and antigenicity.

Sera from HIV-1 infected individuals were examined for their reactivity to the principal neutralizing domain, IGPGRAF sequence, of the V3-loop of HIV-1. Four hybrid proteins carrying this sequence inserted in four different outer loops of a protein that makes up the capsid of an insect virus were used as antigen in a Western blot assay for this survey. All the four antigens showed different activity: sera that recognise all antigens to sera that reacted with only one of them. Competition experiments indicated that the antibodies recognised these proteins with different affinity. Molecular modelling of the hybrid proteins predicted that the inserted sequence adopted different conformations in each position. Comparison of predicted most stable conformations for IGPGRAF indicated that there is a close relationship between conformational similarity to a V3-loop reference structure and the degree of reactivity with sera.

Difference in hepatic uptake of tetra- and di-bromosulfophthalein in rat. Role of hydrophobicity, binding to plasma proteins and affinity for plasma membrane carrier protein.

The relative role of hydrophobicity, binding to plasma proteins and affinity for one of the plasma membrane transport proteins in the hepatic uptake of 3,4,5,6-tetra- (BSP) and 3,6-di- (DBSP) bromosulfophthalein was investigated in the rat. In terms of physicochemical characteristics, the two molecules show different pKa values and degrees of hydrophobicity, as determined from the n-octanol:water partition coefficient. In the intact animal, the plasma clearance and the plasma removal rate after a dose of 1.5 mumol/kg i.v. were significantly (P < 0.001) faster for BSP than DBSP, while no difference was found in the plasma distribution volume. The dissociation constant (Kd) of the high affinity binding sites of plasma proteins also differed for the two anions, being significantly lower for BSP than DBSP (0.95 +/- 0.02 vs 1.44 +/- 0.14 microM, P < 0.001). [35S]BSP uptake by liver plasma membrane vesicles was saturable with an apparent Km of 5.20 +/- 0.80 microM, and was competitively inhibited by DBSP (Ki 18.2 +/- 1.2 microM) indicating a common uptake system. The Kd value for binding of the organic anions to purified bilitranslocase, a plasma membrane protein involved in the electrogenic transport of pthaleins, was also significantly lower for BSP than DBSP (1.10 +/- 0.12 vs 3.02 +/- 0.27 microM, N = 3, P < 0.001), indicating a higher affinity of the former ligand for the carrier protein. No difference was observed in the capacity of the high affinity binding sites (32 +/- 3 vs 33 +/- 3 nmol/mg protein, BSP and DBSP, respectively). These data indicate that BSP and DBSP are two different cholephilic organic anions which share a common uptake mechanism, at least partly mediated by bilitranslocase. The greater affinity of BSP than DBSP for the carrier protein may account for the faster plasma disappearance rate of BSP observed in vivo, in spite of the higher plasma protein binding.

Bilitranslocase and sulfobromophthalein/bilirubin-binding protein are both involved in the hepatic uptake of organic anions.

The hepatic uptake of cholephilic organic anions is a carrier-mediated process. Three distinct proteins [bilitranslocase (BTL), sulfobromophthalein (BSP)/bilirubin-binding protein (BBBP), and organic anion-binding protein] have been isolated from the basolateral plasma-membrane domain of the hepatocyte. To investigate the relative role of the first two of them in accounting for the hepatic uptake of organic anions, we measured the initial rates of uptake of 35S-labeled BSP into rat liver plasma-membrane vesicles. Because transport by BTL is electrogenic but transport by BBBP is electroneutral, studies were done either with or without a positive-inside membrane potential produced by adding valinomycin in the presence of an inwardly directed K+ gradient (outside K+ > inside K+). Both electrogenic and electroneutral transport systems followed saturation kinetics. Electroneutral uptake showed an apparent Km of 20 +/- 3 microM (mean +/- SD) and a Vmax of 1.0 +/- 0.13 nmol.(mg of prot)-1.15 sec-1, whereas the electrogenic portion of BSP uptake exhibited a Km of 5.2 +/- 0.8 microM and a Vmax of 1.1 +/- 0.1 nmol.(mg of prot)-1.15 sec-1. In this case, an overshoot was observed 15 sec after valinomycin addition. Electroneutral BSP uptake was inhibited by incubation with anti-BBBP antibody, whereas anti-BTL antibody did not show any inhibitory effect. Conversely, the electrogenic uptake was inhibited by anti-BTL antibody at a BSP concentration of 5 microM; no inhibition was seen either at 20 microM BSP or upon addition of anti-BBBP antibody. From these data we conclude that the hepatic uptake of organic ions occurs via two immunologically distinct carrier proteins (BTL and BBBP) operating in parallel. BTL is a higher affinity electrogenic transporting system of organic ions, whereas BBBP is a lower affinity electroneutral transporter.

Transformation of fetal secondary cartilage into embryonic bone in organ cultures of human mandibular condyles.

Mandibular condyles of human fetuses, 14-21 weeks in utero, were kept in an organ culture system for up to 60 days. After 6 days in culture, the cartilage of the mandibular condyle appeared to have maintained its inherent structural characteristics, including all its various layers: chondroprogenitor, chondroblastic, and hypertrophic. After 12 days in culture, no chondroblasts could be seen; instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells in the vicinity of the chondroprogenitor zone differentiated into osteoblast-like cells that produced type I collagen. The progenitor cells were still actively incorporating 3H-thymidine. The newly formed osteoid-like tissue lacked both metachromatic reactivity and a response to antibodies against chondroitin sulfate. Instead, the tissue reacted positively for osteocalcin (bone gla-protein). The process of new bone formation further progressed and, by the 20th day in culture, the new bone reacted positively for type I collagen, osteonectin, and to a lesser extent for chondroitin sulfate. The osteoid also underwent mineralization as revealed by both the von Kóssa stain and vital staining with tetracycline. The above feature appeared even more intense in 40-day-old cultures. After 60 days, the newly formed bone contained osteoblasts and osteocytes, whereas the extracellular matrix revealed a high degree of matrix polarization. The results of the present study recapitulate findings reported for organ cultures of mice mandibular condyles. However, the in vitro process of de novo bone formation in human specimens requires a 6-fold longer culture time than that needed for mice condyles.

Influence of acid-secretion blockers on gastric and hepatic alcohol dehydrogenase in rat.

The effects of Cimetidine, Ranitidine, and Omeprazole on gastric and hepatic alcohol-dehydrogenase (ADH) activity was studied in rat. Two apparent values for Km were found for gastric ADH (220 mmol l-1 and 1043 mmol l-1 respectively) and one for hepatic ADH (0.54 mmol l-1). Cimetidine was shown to exert an uncompetitive inhibition of low Km gastric ADH with a Ki of 0.167 mmol l-1 and a competitive inhibition of high Km gastric ADH with a Ki 2.3 mmol l-1. Ranitidine was found to present non-competitive inhibition only on low Km gastric ADH with a Ki of 12 mmol l-1. Omeprazole affects only low Km gastric ADH with a Ki of 5.6 mmol l-1 and presents a linear-mixed type of inhibition. Hepatic ADH was shown to be competitively inhibited only by Cimetidine with a Ki of 6.0 mmol l-1 whereas no inhibition for either Ranitidine and Omeprazole was observed. These results confirm the inhibitory action of Cimetidine on both gastric and hepatic ADH; Ranitidine and Omeprazole show minor effects on ADHS activity and probably on first-pass metabolism.

Carrier-mediated transport of tetrabromosulfonephthalein by rat liver plasma membrane vesicles.

To investigate the molecular requirements and mechanisms for the hepatic uptake of phthaleins, the transport of tetrabromosulfonephthalein (TBS) was investigated in basolateral rat liver plasma membrane vesicles. TBS uptake was electrogenic as greatly accelerated by the creation of a positive-inside membrane potential by the addition of valinomycin in the presence of an inwardly directed potassium gradient. No effect was observed when the ionophore was added in the presence of a sodium gradient. The transport occurred into an osmotic-sensitive space and was saturable with an apparent Michaelis constant of 5.32 +/- 0.56 microM and a maximal velocity of 9.23 +/- 0.25 nmol.s-1.mg protein-1 (mean +/- SD, n = 3 experiments). TBS uptake was directly related to the extra-vesicular pH, indicating the deprotonated quinoid negative-charged form of the dye as the transported species. In contrast, TBS uptake was inversely related to the intravesicular pH, suggesting that protonation inside the vesicles may act as an efficient trap in transport process. Addition of polyclonal monospecific anti-bilitranslocase antibody to liver vesicles specifically inhibited TBS uptake rate (3.27 +/- 0.17 vs. 5.82 +/- 0.61 nmol.s-1.mg protein-1, n = 3, P less than 0.001). These data indicate that TBS is electrogenically transported across the liver cell plasma membrane by bilitranslocase. They also indicate that the presence of a negative charged group on the benzenic ring of the ligand is important in accounting for the transport.

Effect of ethinylestradiol and epomediol on bile flow and biliary lipid composition in rat.

Epomediol (1,3,3-trimethyl-2-oxabicyclo(2.2.2.)octan-6,7-endo,endo-diol) (EPO) is a terpenoid compound shown to reverse 17 alpha-ethinylestradiol (EE)-induced cholestasis in rat. The effect is related to the restoration of normal liver plasma membrane fluidity values. To further characterize the effect of EPO, bile flow and biliary lipid composition were measured in rats treated either with EE or EE associated with EPO. EE significantly reduced the bile flow; this reduction was prevented by concomitant treatment with EPO with an increase in the bile salt secretion rate. EPO alone showed a choleretic effect. The biliary secretion rate of cholesterol was also significantly reduced by EE while being comparable to controls in EE-EPO-treated animals. Phospholipid (PL) biliary excretion was significantly (P less than 0.002) increased by EE either alone or combined with EPO. After EE treatment, the biliary PL composition showed a reduction in phosphatidylcholine (PC) concentration with a parallel increase in lyso-phosphatidylcholine (LPC) when compared to control animals (PC:LPC ratio 5.0 +/- 2.5 vs 26.8 +/- 9.9, mean +/- SD, P less than 0.005). EPO administration to EE-treated rats restored the biliary PC:LPC ratio to control values (27.6 +/- 10.6). EPO alone did not show any appreciable effect as compared to both control and EE-EPO treated animals. As increased concentrations of LPC have been reported to induce an alteration in the function of membrane lipids and membrane-associated proteins, such as regulatory enzymes for bile acid, cholesterol and phospholipid metabolism, these results suggest that the protective effect of EPO in EE-induced cholestasis may be related to the reversal of the alterations in membrane lipid composition and function induced by EE.

Transport of sulfobromophthalein and taurocholate in the HepG2 cell line in relation to the expression of membrane carrier proteins.

The transport of two different classes of organic anions (cholephilic dyes; the sulfobromophthalein, BSP, and bile acids; taurocholate, TC) was investigated in the HepG2 cell line. At 37 degrees C, BSP uptake was found to be biphasic with an apparent saturative curve in the concentration range between 0-6 microM followed by a linear component up to 18 microM. Kinetic constant determination showed an apparent Km of 26.6 +/- 3.1 microM and a Vmax of 5.64 +/- 0.82 nmol BSP.min-1.mg prot-1. At 4 degrees C, uptake was linear. By subtracting this latter component from the total uptake, a saturable, carrier mediated uptake was found with an apparent Km of 3.6 +/- 1.0 microM BSP and a Vmax of 0.37 +/- 0.04 nmol BSP.min-1.mg prot-1 (m +/- SEM, n = 6). These values were fully comparable with those found in freshly isolated male hepatocyte. Immunoblot analysis of HepG2 cell plasma membrane revealed the presence of bilitranslocase when tested against a monospecific antibody against this carrier molecule. On the contrary, TC uptake was linear up to concentration of 100 microM TC. No difference was observed in the presence or absence of Na+. Immunoprecipitation analysis showed the absence of the putative carrier of TC. These data indicate that the HepG2 cell line expresses a functioning bilitranslocase-mediated system. Conversely, carrier mediated bile acid uptake is absent in line with the lack of expression of the carrier protein.

Immunohistochemical studies of the extracellular matrix in the condylar cartilage of the human fetal mandible: collagens and noncollagenous proteins.

This study provides data concerning the cells and their extracellular matrix in prenatal human mandibular condylar cartilage. The latter cartilage represents a secondary type of cartilage since it develops late in the morphogenesis of the craniofacial skeleton. The cartilage of the mandibular condyle is actively involved in endochondral ossification, thus showing all the phases of cartilage growth, maturation, and mineralization that precedes de novo bone formation. The present study focused on the localization and distribution of the major macromolecules that are normally encountered in cartilage and bone, including collagens, proteoglycans, fibronectin, osteonectin, osteocalcin, alkaline phosphatase, and anchorin CII. It became clear that the mineralized zone of the cartilage already contained bone-specific antigens; thus the above zone might serve as an essential propagative predecessor in the ossification process.

Monoclonal antibodies define structural alterations of the thyroglobulin secreted by well-differentiated thyroid carcinomas.

We have studied the sorting and alteration of the thyroglobulin (tg) secreted by well-differentiated carcinomas using a group of anti-tg monoclonal antibodies. A previously described monoclonal antibody, specific for the hormogenic iodinated epitopes, shows that in carcinomas the iodinated tg is not secreted in the colloid as in normal glands but is accumulated in the cytoplasm of the cells. Separation of tg in a concanavalin A column, to isolate the correctly glycosilated protein, reveals that hormogenesis is present only in glycosilated tg with a correct final configuration. An other monoclonal antibody, specific for an epitope connected with the carbohydrate moiety of the tg, reacts with tgs from normal glands and from any thyroid lesion except tg from carcinomas. This tg of carcinomas presents an alteration in the oligosaccharide chains that can be detected by this antibody. The alteration is specific for malignant tumors and could be connected with the missing exocytosis of the iodinated tg in carcinomas.

Bilitranslocase localization and function in basolateral plasma membrane of renal proximal tubule in rat.

Bilirubin and phthalein dyes are taken up by the liver via a carrier-mediated mechanism operated at least in part by bilitranslocase (BTL). Because they also undergo renal transport, the presence and function of BTL was investigated in rat renal tubular plasma membrane vesicles. Transport of sulfobromophthalein (BSP) was enriched in basolateral domain of plasma membrane and followed the distribution pattern of Na(+)-K(+)-ATPase but not of gamma-glutamyltransferase. BSP uptake was inhibited by addition of monospecific antibodies raised against hepatic BTL. As in liver vesicles, BSP transport was electrogenic, being greatly accelerated by addition of valinomycin in presence of an inwardly directed K+ gradient. Apparent Km of BSP transport was 17 +/- 2 microM (n = 3 expts), one order of magnitude higher than that measured in liver; however, Vmax was similar to that described in liver vesicles (429 +/- 18 nmol BSP.mg protein-1.min-1, n = 3 expts). Competitive inhibition was observed with both unconjugated bilirubin (Ki, 2.9 +/- 0.2 microM) and rifamycin SV (Ki, 76 +/- 10 microM), known competitors for hepatic BTL-mediated transport of BSP. Immunoblotting studies with anti-BTL monospecific antibodies revealed presence of a single positive band only in basolateral-enriched membrane fraction; its apparent molecular mass was 37 kDa, virtually identical to that of hepatic protein. Immunohistochemistry confined presence of BTL to renal proximal tubules (RPT) We conclude that BTL is present in basolateral plasma membrane of RPT cells. Lower affinity of renal, compared with hepatic protein, for substrates might explain the marginal role of kidney in plasma clearance of bilirubin and cholephilic dyes.

Reconstitution of sulfobromophthalein transport in erythrocyte membranes induced by bilitranslocase.

Bilitranslocase, the protein responsible for the anion translocation at the sinusoidal plasma membrane level in liver, was shown to be able to reconstitute the transport of sulfobromophthalein in liposomes in the past. The protein preparation used in those experiments consisted of two subunits of 35.5 and 37 kDa. The isolated 37 kDa protein, when inserted in erythrocyte membrane vesicles, confers to the particles the ability to carry out an electrogenic transport of sulfobromophthalein. The effect is specific and can be inhibited by monospecific polyclonal antibodies raised against the protein. In may be concluded that the 37 kDa protein band, present in previous preparations of bilitranslocase, is not only a necessary but also a sufficient component of the transport system for bilirubin and functional analogues.

Reversal of ethinylestradiol-induced cholestasis by epomediol in rat. The role of liver plasma-membrane fluidity.

Epomediol (EPO) is a synthetic terpenoid compound shown to be active in increasing bile flow and some enzymatic activities of liver plasma membranes in the rat. The possible effect of EPO treatment in the ethinyl-estradiol (EE) induced cholestasis in the rat was investigated by measuring the hepatic transport of sulfobromophthalein (BSP) (plasma clearance and biliary secretion) and bile flow. Liver plasma membrane fluidity was also determined by the steady state fluorescence polarization (P) of diphenylhexatriene (DPH). EE administration (5 mg/kg s.c. for 5 days) was followed by a significant, comparable reduction (P less than 0.001) in BSP plasma clearance and biliary excretion and in bile flow. Intraperitoneal administration of EPO (100 mg/kg) to EE-treated rats restored both parameters of BSP transport, as well as bile flow, to control values. Liver plasma membrane fluidity was markedly (P less than 0.01) decreased by EE administration with a concomitant reduction (P less than 0.01) in Na+/K+-ATPase activity. EPO administration significantly increased membrane fluidity to values higher either to cholestatic (P less than 0.05) or control (P less than 0.05) animals. On the contrary, EPO did not influence Na+/K+-ATPase activity in either EE-treated or control animals. These data indicate that EPO fully reverses the impairments of BSP transport and bile flow induced by EE, possibly by reversing the decrease in liver plasma membrane fluidity induced by the synthetic estrogen. On the contrary, the EE-mediated decrease in Na+/K+-ATPase activity was not reversed by EPO.

Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver: immunochemical evidence using mono- and poly-clonal antibodies.

Monoclonal antibodies raised against bilitranslocase, may display either inhibitory or enhancing activity on the electrogenic transport of sulfobromophthalein, evoked in rat liver plasma-membrane vesicles by the addition of valinomycin in the presence of K+. In both cases, the target protein is identified with a 37 kDa band in SDS-mercaptoethanol gel electrophoresis of solubilized membranes. The electrophoretically homogeneous protein isolated by ion-exchange chromatography, corresponds in all respects to the 37 kDa protein band of bilitranslocase, obtained in the past by different techniques. Using this protein as antigen, a polyclonal monospecific antibody preparation has been obtained. As expected, the antibody preparation inhibits the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver. It is concluded that the 37 kDa protein of bilitranslocase is at least a necessary component of the transport system involved in the sulfobromophthalein movement in plasma membrane.