Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus.

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.

Identification of a cis-acting element and a novel trans-acting factor of the glutamine synthetase gene in liver cells.

In the mammalian liver the expression of the enzyme glutamine synthetase (GS) is restricted to a small population of hepatocytes. In cells expressing the enzyme up to 3.5% of total cellular protein is GS. In order to identify enhancer elements contributing to this extraordinarily high level of expression we focused on a region roughly 2.5 kbp upstream of the GS promoter. Gel mobility shift assays revealed binding of an unknown protein within the most distal part of this region and reportergene assays demonstrated that roughly 60 bp downstream from position -2503 are indispensable for protein binding and the full effect of the enhancer. In UV cross-link analysis a 38 kDa nuclear protein that binds to the sequence was identified in rat hepatocytes. This nuclear protein, designated as upstream binding factor of the GS gene (UFGS) seems to play an important role in high-level expression of GS in liver.

Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.

Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.

Transfection of normal human epidermal keratinocytes with lipid/dna complexes in vitro.

Highly proliferative normal human epidermal keratinocytes (NHK) were isolated from human foreskin biopsies, cultivated in serum-free medium and characterized by flow cytometry. The expression of cytokeratin 19, cytokeratin 14 and vimentin indicated that the suspension contained a high percentage of undifferentiated cells of the basal epidermal layer. The NHK were transfected in vitro with lipid/DNA complexes made of Effectene or Lipofectamine and different reporter genes. The transfection efficiency of Effectene/DNA complexes was 20fold higher compared to Lipofectamine. Transfected keratinocytes continued to grow and developed within 2 weeks a cellular multilayer (3-D culture). Areas of transfected cells were detected within this layer.

Heterogeneous expression of mRNA in rat liver lobules as detected by differential display.

Although liver hepatocytes appear to be uniform histologically, they are considerably heterogeneous with respect to their individual physiological capacities. In order to find still unknown genes that are heterogeneously expressed and with the aim of evaluating the usefulness of the differential display technique for this purpose, we performed differential displays with mRNA isolated from hepatocytes from the periportal and pericentral zone of the rat liver. In this way we identified at least two mRNAs exclusively expressed in the pericentral fraction. Sequence analysis revealed that the corresponding genes encode proteins with proline-glutamate dipeptide repeats similar to ones previously identified in rat pheochromocytoma and brain. In situ hybridization confirmed the heterogeneous distribution of the mRNA. Only one to two cell lines surrounding the terminal hepatic venules were positive, strongly resembling the heterogeneous expression of the enzyme glutamine synthetase. Our work demonstrates that the differential display method is a useful tool for the identification of genes that are differentially expressed in individual parenchymal cells. In fact, our results prove that differential display technology can be used for the identification of cellular markers for distinct subpopulations of cells in a given tissue.

Post-transcriptional inhibition of glutamine synthetase induction in rat liver epithelial cells exerted by conditioned medium from rat hepatocytes.

Recently, a soluble factor produced by primary periportal hepatocytes and different from glutamine was found to completely block induction of glutamine synthetase (GS) by dexamethasone (DEX) in the liver cell line RL-ET-1G. Using Northern and Western blotting we investigated whether this block is regulated on the transcriptional or the post-transcriptional level. Three different species of GS mRNA were detected that all increased (though in different proportions), when the cells were exposed to DEX for 24 h. Further maintenance for another 24 h in normal DEX-containing culture medium, conditioned medium from primary periportal hepatocytes or medium containing glutamine did not result in significant differences in GS mRNA levels. In contrast, GS protein and specific GS-activity remained high only under normal culture conditions, whereas both returned to basal levels in conditioned and glutamine-supplemented culture medium. Throughout, GS protein content and specific GS-activity strongly correlated (r = 0.98) excluding that GS-activity is regulated by chemical modification. These data suggest that the decrease in enzyme activity caused by cultivation in CM is controlled on the post-transcriptional level.

Treatment of cirrhotic rats with L-ornithine-L-aspartate enhances urea synthesis and lowers serum ammonia levels.

CCl4-induced cirrhosis of rats was used for studying the influence of L-ornithine-L-aspartate (OA) on hyperammonemia. OA given to cirrhotic rats (2 g/kg daily) for 2 wk slightly increased net body weight and led to a significant increase in plasma urea levels and a decrease in plasma ammonia levels. Serum concentrations of glutamate, glutamine and arginine decreased significantly. In the livers of the OA-treated rats the activities of carbamoylphosphate synthetase I and arginase increased by 30 and 40%, respectively, approaching normal levels. No change in the activities of the other urea cycle enzymes as well as of glutamate dehydrogenase, glutaminase and glutamine synthetase was found. The negative correlation between glutamine synthetase activity and plasma ammonia levels reported previously for cirrhotic rats (Gebhardt and Reichen, Hepatology 20:684-691, 1994) was corroborated for cirrhotic animals not treated with OA, but was no longer apparent in OA-treated cirrhotic rats. Despite this improvement, plasma ammonia levels still varied considerably reflecting the variable accessibility and activities of glutamine synthetase in cirrhotics. Cultured hepatocytes from the two groups of rats showed a similar stimulation of urea production by addition of ammoniumacetate and/or OA to Hanks' buffered salt solution. In Williams medium E, however, the hepatocytes from the OA group produced significantly more urea than those from controls. These results suggest that treatment of cirrhotic rats with OA considerably improves urea production favoring the detoxification of ammonia that, however, is still limited by the severe alterations in liver architecture that are not influenced by OA in a 2-wk period.

Cell-cell interactions in the regulation of the expression of hepatic enzymes.

The expression of enzymes and other proteins in liver parenchyma is heterogeneous and shows distinct features depending on whether all or only a subset of hepatocytes are involved. Recent advances in the elucidation of the factors that control zonated expression have provided new insights into the regulatory mechanisms that might underlie the different patterns of heterogeneity. While humoral factors seem to play a predominant role in the dynamic adaptation of zonated expression, newly identified intrahepatic (zonation) factors might be responsible for a static dissection of the parenchyma into different expression compartments. These different principles for determining heterogeneous enzyme expression are presented and discussed for cholesterol 7 alpha-hydroxylase and glutamine synthetase.

Cis-regulatory sequences from the first intron of the rat glutamine synthetase gene are involved in hepatocyte specific expression of the enzyme.

In order to identify regulatory elements involved in the hepatocyte specific expression of the enzyme glutamine synthetase [GS (E.C. 6.3.1.2)] we analyzed the first intron of the rat GS gene. A sequence analysis detected clusters of potential transcription factor binding sites in regions that are hypersensitive for DNase I, including sites for Sp1, HNF3 and elements related to binding of members from the C/EBP family. By use of DNA fragments with putative regulatory elements, reporter genes have been constructed that were transfected into isolated hepatocytes in primary culture and into HepG2 hepatoblastoma cells. By these experiments we cold show that sequences from the first intron are able to enhance transcription specifically in hepatocytes but not in cells from the hepatoblastoma cell line. The existence of enhancer effects in the first intron of the GS gene and their restriction to hepatocytes demonstrates that aside from regulatory regions upstream of the transcription start point, there are also downstream regions involved in the specific expression of the gene. We conclude that intronic elements are involved in the pretranslational regulation of the expression of the GS as part of a complex interplay between different regions of the gene.

Transient transfection of primary cultured hepatocytes using CaPO4/DNA precipitation.

We present a detailed protocol for the transient transfection of non-proliferating primary cultured hepatocytes that is easily reproducible. Using a modification of the classical CaPO4/DNA precipitation method, this protocol is an inexpensive alternative to other methods that are often cumbersome, expensive, difficult to reproduce or harmful to primary hepatocytes. Because only 0.5 x 10(6) cells are needed for a single transfection experiment, several reporter genes can be introduced into hepatocytes of a single liver preparation. With our protocol, different plasmids can be introduced into one cell. In this way, cis-trans interactions can be examined and reporter gene expression can be normalized for transfection efficiency. Furthermore, we describe details of a transfection experiment with two different reporter gene vectors using a luciferase gene and a lacZ gene. The results presented may be helpful to other groups concerned with improved timing of transfection experiments.

Heterogeneous (positional) expression of hepatic glutamine synthetase: features, regulation and implications for hepatocarcinogenesis.

Glutamine synthetase expression in liver parenchyma is restricted to a small population of pericentral hepatocytes surrounding the central veins. Studies on the development of this heterogeneous (positional) gene expression and of the changes observed in response to experimental alterations of liver physiology or manipulations of hepatocytes in culture have revealed that it is dependent on cell-cell and cell-matrix interactions rather than on the levels of hormones and other modulating factors. The considerable stability of GS expression may point to further events leading to a defined differentiated GS+ phenotype. Observations during experimental hepatocarcinogenesis indicate that strong GS expression may be used for tracing hepatocellular lineages during preneoplastic and early neoplastic stages. Furthermore, these studies suggest a relationship between the GS+ phenotype and enhanced growth of these lesions. Future studies should help to define the diagnostic value of GS and its significance for new chemotherapeutic strategies.

Primary structure of a gene-sized DNA encoding calmodulin from the hypotrichous ciliate Stylonychia lemnae.

We have isolated and characterized a gene-sized DNA encoding calmodulin (Clm) from macronuclear (MA) DNA of the hypotrichous ciliate, Stylonychia lemnae. The gene has 3500 copies per macronucleus. The length of the gene was deduced by agarose-gel electrophoresis of MA DNA and Southern blot analysis using a Clm cDNA probe from chicken. We then isolated the gene from a MA library. The overall length of the gene is 821 bp with a 450-bp intronless coding region. The deduced amino acid (aa) sequence of ciliate Clm has 149 aa and an M(r) of 16,819. Both ends of the cloned gene have the hypotrichous telomeric C4A4 repeat. The coding region is flanked by a 158-bp 5'-leader sequence and a 3'-trailer sequence of 213 bp. S1 analysis was used to locate the transcription start point (tsp) 49 bp upstream from the start codon. No common eukaryotic transcription signals were found upstream from the tsp. A second gene-sized DNA, detected by its cross-hybridization with the Clm DNA, predicts the existence of a second Ca(2+)-binding protein with only one Ca(2+)-binding site. It's function and biological significance is yet unknown.