Selective stimulation of the JAK/STAT signaling pathway by silica nanoparticles in human endothelial cells.

Silica nanoparticles (SiNP) are frequently used in pharmaceutical formulations. Intravenously administered, these particles are in close contact with the vascular endothelium. However, preliminary safety assessments of these novel excipients have indicated that SiNP are potentially cytotoxic and can trigger inflammation. In order to elucidate mechanisms of SiNP mediated inflammation, cerebral microvascular endothelial cells and primary umbilical endothelial cells were incubated with SiNP at doses between 10ng/ml and 250µg/ml. Two types of 110nm SiNP with different surface charge were synthesized and characterized. Uptake, cell viability, apoptosis, necrosis, oxidative stress, as well as interferences with both JAK/STAT and NF-κB pathways were studied. SiNP uptake leads to a cell viability decrease and promotes generation of reactive oxygen species (ROS) in a time- and dose-dependent manner. Furthermore, SiNP are able to trigger the activation of the STAT1 pathway. In contrast, no significant activation of STAT3, STAT6 or NF-κB could be detected. Additionally, modulation of the major histocompatibility complex (MHC) class I proteins was observed for cationic SiNP at low doses. Our results show the potential of SiNP to trigger selective activation of inflammatory signaling pathways in endothelial cells and thereby contribute to a better understanding of the toxicological profile of SiNP.

[Locoregional treatments of brain metastases for patients with metastatic cutaneous melanoma: French national guidelines]. / Traitements locorégionaux de métastase(s) cérébrale(s) chez des patients atteints d'un mélanome cutané métastatique : recommandations nationales françaises.

INTRODUCTION: The management of metastatic cutaneous melanoma is changing, marked by innovative therapies. However, their respective use and place in the therapeutic strategy continue to be debated by healthcare professionals. OBJECTIVE: The French national cancer institute has led a national clinical practice guideline project since 2008. It has carried out a review of these modalities of treatment and established recommendations. METHODS: The clinical practice guidelines development process is based on systematic literature review and critical appraisal by experts. The recommendations are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines are reviewed by independent practitioners in cancer care delivery. RESULTS: This article presents the results of bibliographic search, the conclusions of the literature and the recommendations concerning locoregional treatments of brain metastases for patients with metastatic cutaneous melanoma.

[Management of patients with metastatic cutaneous melanoma: French national guidelines. French National Cancer Institute]. / Prise en charge thérapeutique des patients atteints d'un mélanome cutané métastatique : recommandations nationales françaises. L'Institut national du cancer.

BACKGROUND: Recent years have seen the emergence of new molecules for the treatment of patients with metastatic cutaneous melanoma, with significant benefits in terms of survival and the opening of new therapeutic perspectives. In addition, many techniques are currently being developed for locoregional treatment of metastatic sites. Management of metastatic melanoma is thus fast-changing and is marked by innovative therapeutic approaches. However, the availability of these new treatments has prompted debate among healthcare professionals concerning their use and their place in therapeutic strategy. AIMS: Since 2008, the French National Cancer Institute (INCa) has been leading a project to define and diffuse national clinical practice guidelines. It has performed a review of these treatment methods, which it aims to circulate, and it is seeking to develop recommendations in order to allow nationwide implementation of innovative approaches while promoting good use thereof. METHODS: The clinical practice guidelines development process is based on systematic literature review and critical appraisal by experts within a multidisciplinary working group, with feedback from specialists in cancer care delivery. The recommendations are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines are reviewed by independent practitioners in cancer care delivery. RESULTS: This article presents the national recommendations for first- and second-line systemic treatment and for locoregional treatment of metastatic sites in patients presenting metastatic cutaneous melanoma.

hH-Rev107, a class II tumor suppressor gene, is expressed by post-meiotic testicular germ cells and CIS cells but not by human testicular germ cell tumors.

By systematic analysis of a human testis library, we have isolated the hH-Rev107-3 cDNA, identical to hH-Rev107-1 cDNA, which was previously described as a class II tumor suppressor gene. In this study, two transcripts (1 and 0.8 kb) were detected by Northern blot in all human tissues, excepted in thymus. The strongest expression was found in testis, skeletal muscle and heart. These two mRNA are probably transcribed from only one gene that we mapped to the q12-q13 region of the chromosome 11. In human testis, hH-Rev107 gene expression was localized, by in situ hybridization, within the round spermatids. To investigate a possible role for hH-Rev107 protein in testicular malignant growth, we examined the expression of this gene in germ cell tumors. A strong hH-Rev107 gene expression was observed in normal testis as well as in samples with preinvasive carcinoma in situ but was completely absent in overt tumors, both seminomas and non-seminomas. By in situ hybridization, CIS was found hH-Rev107 positive and tumor negative. A semi-quantitative assessment of hH-Rev107 mRNA level in testicular germ cell tumors, by RT-PCR, exhibited a ninefold decrease in the gene expression. No gross structural aberrations of hH-Rev107 gene were detected in these human primary tumors. The results suggest that down-regulation of hH-Rev107 may be associated with invasive progression of testicular germ cell tumors.

Two muscle-specific LIM proteins in Drosophila.

The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.

High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.

The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.

Human testis specifically expresses a homologue of the rodent T lymphocytes RT6 mRNA.

A human homologue of the rodent T cell mono ADP-ribosyl transferase RT6 mRNA was identified by a systematic analysis of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono ADP-ribosyl transferase and a C-terminal part characteristic of glycophosphatidyl inositol anchored protein. This mRNA, transcribed from a gene localized in 4q13-q21, is not expressed in white blood cells but is specific for human testis in which it is likely to correspond to a new ADP-ribosyl transferase.

Tissue- and developmental-stage-specific methylation in the two kidney promoters of the rat gamma-glutamyl transpeptidase gene.

We have investigated, using DNA methylation patterning, the site-specific methylation of promoters I and II of the rat gamma-glutamyl transpeptidase gene. This analysis was done in fetal, newborn and adult rat kidney, in which promoters I and II are progressively active during development, as well as in rat liver, which never expresses mRNAs from these two promoters. During kidney development, a progressive demethylation occurs in the promoter I and II region, specially at the level of the most proximal MspI site of promoter II. A progressive reorganization of the methylated sites within the 5' end of the gene also occurs during liver development.

Repetitive 5-azacytidine treatments of Fao cells induce a stable and strong expression of gamma-glutamyl transpeptidase.

The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.

DNA methylation patterns of the rat gamma-glutamyl transpeptidase gene in embryonic, adult and neoplastic liver.

The methylation status of the rat gamma-glutamyl transpeptidase (GGT) gene was investigated during liver development and hepatocarcinogenesis. The analysis with the restriction enzymes MspI/HpaII revealed that, during ontogeny, there is a progressive methylation of the GGT gene that coincides with a progressive decrease in GGT activity. Thus, there is an inverse correlation between methylation and expression of the GGT gene, suggesting a role for DNA methylation in the regulation of the gene during normal differentiation. The methylation patterns of the GGT gene in liver tumours induced by aflatoxin B1 exhibit heterogeneity. Nevertheless, a band of 5.7 kb was observed in all the DNA samples from aflatoxin B1-induced tumours which was not present in control liver DNA. The specificity of the DNA methylation changes was assessed using nafenopin, which induces hepatic tumours without elevation of GGT activity. We conclude that, during hepatocarcinogenesis, there is a modification of the DNA methylation pattern of the GGT gene, but there is no simple correlation with GGT activity. In no case was the GGT gene methylation in hepatocarcinogenesis found to be equivalent to the pattern observed in fetal liver. Thus if methylation is involved in the regulation of GGT gene transcription, the mechanisms must be different in fetal liver and hepatocarcinoma.

Localization by in situ hybridization of gamma-glutamyl transpeptidase mRNA in the rat kidney using 35S-labeled RNA probes.

gamma-Glutamyl transpeptidase (GGT) is involved in the extraction of plasma glutathione in the postglomerular compartment of the kidney. The enzyme is distributed in the proximal tubule associated with the apical brush border. Using in situ hybridization with 35S-labeled anti-sense and sense RNA GGT probes on rat kidney cryostat sections, the present study demonstrates that GGT mRNA transcripts are detected in proximal tubules localized in the inner cortex, outer medulla, and medullary rays of the cortex. Such a distribution suggests that the GGT gene is mainly expressed in the more distal part of the proximal tubule, i.e., the pars recta. As a control, in situ hybridization has also been performed using a 35S-labeled beta-actin anti-sense RNA probe showing a diffuse pattern of distribution of beta-actin RNA transcripts particularly in the outer cortex. This highly sensitive method using single strand asymetric RNA probes, with a markedly reduced nonspecific binding is promising for the study of gene expression patterns in the kidney in order to clarify their heterogeneity in the various segments of the nephron.

Regulation of mouse mammary-gland gamma-glutamyltranspeptidase mRNA during pregnancy, lactation and weaning.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.

Mechanism of gamma-glutamyl transpeptidase release in serum during intrahepatic and extrahepatic cholestasis in the rat: a histochemical, biochemical and molecular approach.

The mechanism of the elevation of serum gamma-glutamyl transpeptidase activity in cholestasis is not clear. We therefore analyzed rat gamma-glutamyl transpeptidase activities in liver, bile and serum during intrahepatic cholestasis induced by a single dose of alpha-naphthyl isothiocyanate (20 mg/100 gm body weight) and during extrahepatic cholestasis after bile duct ligation. At days 1 and 2 after alpha-naphthyl isothiocyanate ingestion, we saw a fivefold and a 60-fold increase in serum and bile gamma-glutamyl transpeptidase activities, respectively. These increases were associated with a decrease in hepatic gamma-glutamyl transpeptidase activity and of corresponding mRNA. Simultaneously, necrosis of the biliary epithelium appeared in portal tracts. From day 2 to day 14, gamma-glutamyl transpeptidase activity in bile and serum progressively returned to basal levels; in the liver, cholangiolar proliferation was mild and was associated with moderate elevation of the gamma-glutamyl transpeptidase activity and of its corresponding mRNA. In extrahepatic cholestasis, a 10-fold increase in serum gamma-glutamyl transpeptidase activity was detected between day 0 and day 14. This increase was associated with major cholangiolar proliferation and with a progressive rise in hepatic gamma-glutamyl transpeptidase activity and in specific mRNA; in bile, gamma-glutamyl transpeptidase activity was slightly elevated. In these two models of cholestasis, histochemically detected gamma-glutamyl transpeptidase activity was largely predominant in biliary cells. We found no significant induction of gamma-glutamyl transpeptidase activity in hepatocytes. These results suggest that in these two models of cholestasis, the increase in serum gamma-glutamyl transpeptidase activity is of biliary cell origin and does not originate from hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in human tissues.

Human gamma-glutamyl transpeptidase (GGT)1 is composed of two subunits derived from a single precursor (Nash, B., and Tate, S.S. (1984) J. Biol. Chem. 259, 678-685; Finidori, J., Laperche, Y., Tsapis, R., Barouki, R., Guellaën, G., and Hanoune, J. (1984) J. Biol. Chem. 259, 4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M.N., Aggerbeck, M., Hanoune, J., and Guellaën, G. (1986) Proc Natl. Acad. Sci. U.S.A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3'-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, placenta, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.

Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney.

In human, the two subunits of gamma-glutamyl transpeptidase (GGT) arise from a common precursor encoded by a multigene family. Until now, a single specific coding sequence for this precursor (type I) has been identified in human placenta and liver. In the present study, we have isolated from a human kidney cDNA library, a GGT specific clone (0.8 Kb). The sequence of which (type II) i) covers the carboxy terminal part of the GGT precursor, ii) exhibits 22 point mutations and a 30 bp deletion as compared to the type I GGT sequence. The sequencing of a human genomic clone reveals that this type II GGT mRNA is encoded by a different gene than the type I GGT mRNA. Both type I and type II GGT mRNAs are expressed in human liver, while almost exclusively type II GGT mRNA is detected in human kidney.

Effects of hCGRP I and II on gastric blood flow and acid secretion in anesthetized rabbits.

Effects of intravenously administered human calcitonin gene-related peptides (hCGRP) I and II on regional blood flow and gastric acid secretion were examined in barbiturate-anesthetized rabbits. Blood flow was measured by injection of radioactively labeled microspheres at 0, 10, 20, 30, and 60 min. hCGRP I and II and vehicle were infused intravenously in five rabbits in rising doses of 0.01 (0-10th min), 0.03 (11-20th min), and 0.1 microgram.kg-1.min-1 (21-30th min). hCGRP I and II increased gastric blood flow dose dependently. Moreover, hCGRP I raised regional conductance (inverse of vascular resistance) in the stomach, duodenum, heart, brain, and skeletal muscle. As a result of the increased total peripheral conductance the mean arterial pressure was reduced, but the cardiac output remained unchanged. hCGRP II increased blood flow and conductance selectively in the stomach and the pancreas. The total peripheral conductance and mean arterial pressure remained unchanged. Apparently, hCGRP II exerts a more localized effect on the stomach than hCGRP I. hCGRP I and II did not affect basal gastric acid secretion. Pentagastrin-stimulated acid secretion was increased by 28% with hCGRP I (0.025 micrograms.kg-1.min-1) and decreased by 27% with hCGRP II (0.025 micrograms.kg-1.min-1). The inverse effect of hCGRP I and II and the parallel stimulation of blood flow brought about with hCGRP I and II indicate a different mode of action of the peptides on gastric blood flow and gastric acid secretion.

gamma-Glutamyl transpeptidase: a single copy gene in the rat and a multigene family in the human genome.

gamma-Glutamyl transpeptidase (GGT) genomic sequences were isolated from rat and human libraries using a rat GGT cDNA as a cross-species hybridization probe. Characterization of the human GGT clones by restriction mapping clearly establishes that at least four different GGT genes or pseudogenes are present in the human genome. All the rat genomic clones cover a 12.5-kilobase sequence and exhibit a unique restriction pattern. A precise quantitation of the rat GGT gene copy number by Southern blot analysis demonstrates that this sequence is present as a single copy/rat haploid genome. Therefore, the GGT gene organization is different between rat and human species; this raises the possibility of different regulatory mechanisms in the two species.

Assignment of the human gamma-glutamyl transferase gene to the long arm of chromosome 22.

We have determined the chromosomal location of the human gene for gamma-glutamyltransferase (GGT). This study was done by in situ hybridization of human metaphase spreads with a rat cDNA probe specific for this enzyme and constructed from two clones previously characterized in our laboratory. The final construct had a 1.6-kb-long insert covering 92% of the coding sequence for GGT. The new insert was also freed of any GC tails introduced for the cDNA cloning, because we observed that these sequences were responsible for a high background. Using this probe for the analysis of 136 human metaphase spreads, we observed a strong specific signal on chromosome 22 at the interface of q111-112 and a minor peak in q131. Thus GGT might represent a new marker for the study of certain diseases which have chromosomal abnormalities at these loci.

About the specificity of photoinduced affinity labeling of Escherichia coli ribosomes by dihydrorosaramicin, a macrolide related to erythromycin.

Photoactivation of the [3H]dihydrorosaramicin chromophore at a wavelength above 300 nm allows the covalent attachment of the macrolide antibiotic to the bacterial ribosome. Bidimensional electrophoresis shows that the radioactivity is mainly associated with proteins L1, L5, L6, L15, L18, L19, S1, S3, S4, S5 and S9. When photoincorporation of the drug is conducted in the presence of puromycin as effector of [3H]dihydrorosaramicin-binding sites, a decrease in the labeling of most proteins is observed, except for L18 and L19, which are radiolabeled to a larger extent. These results allow us to speculate that L18 and L19 belong to the high-affinity binding site of rosaramicin antibiotic.

Effect of P and A site substrates on the binding of a macrolide to ribosomes. Analysis of the puromycin-induced stimulation.

The puromycin-induced stimulation of [3H]dihydrorosaramicin binding is due to a twofold increase in affinity of the macrolide antibiotic, with no change in the number of binding sites. Conversely, the binding of [3H]puromycin (A site) is stimulated by rosaramicin. The synergistic effect observed between the two antibiotics can be explained by a conformational change with positive effect, which occurs at the level of their binding sites. Various effectors of [3H]dihydrorosaramicin binding have been tested. Adenosine and dimethyladenosine stimulate the binding; phenylalanine, uridine and gougerotin (A site) have no effect whereas AMP, ADP, ATP, GTP, puromycin 5'-phosphate and lincomycin (P site) are inhibitors. These results point to the importance of the purine moiety in the stimulatory effect and of the phosphate function in reversing this effect. It is concluded that rosaramicin binds to the ribosomal P site and that the synergism observed between rosaramicin and puromycin may be related to interactions between the A and P sites.