Pulmonary and hepatic effects after low dose exposure to nanosilver: Early and long-lasting histological and ultrastructural alterations in rat.

Although environmental airborne silver nanoparticles (AgNPs) levels in occupational and environmental settings are harmful to humans, the precise toxic effects at the portal entry of exposure and after translocation to distant organs are still to be deeply clarified. To this aim, the present study assessed histopathological and ultrastructural alterations (by means of H&E and TEM, respectively) in rat lung and liver, 7 and 28 days after a single intratracheal instillation (i.t) of a low AgNP dose (50 microg/rat), compared to those induced by an equivalent dose of ionic silver (7 microg AgNO3/rat). Lung parenchyma injury was observed acutely after either AgNPs or AgNO3, with the latter compound causing more pronounced effects. Specifically, alveolar collapse accompanied by inflammatory alterations and parenchymal fibrosis were revealed. These effects lasted until the 28th day, a partial pulmonary structure recovery occurred, nevertheless a persistence of slight inflammatory/fibrotic response and apoptotic phenomena were still detected after AgNPs and AgNO3, respectively. Concerning the liver, a diffuse hepatocyte injury was observed, characterized by cytoplasmic damage and dilation of sinusoids, engulfed by degraded material, paralleled by inflammation onset. These effects already detectable at day 7, persisting at the 28th day with some attenuations, were more marked after AgNO3 compared to AgNPs, with the latter able to induce a ductular reaction. Altogether the present findings indicate toxic effects induced by AgNPs both at the portal entry (i.e. lung) and distant tissue (i.e. liver), although the overall pulmonary damage were more striking compared to the hepatic outcomes.

Dipeptidylpeptidase-IV activity and expression reveal decreased damage to the intrahepatic biliary tree in fatty livers submitted to subnormothermic machine-perfusion respect to conventional cold storage.

Graft steatosis is a risk factor for poor initial function after liver transplantation. Biliary complications are frequent even after normal liver transplantation. A subnormothermic machine perfusion (MP20) preservation procedure was developed by our group with high potential for reducing injury to hepatocytes and sinusoidal cells of lean and fatty livers respect to conventional cold storage (CS). We report the response of the biliary tree to CS or MP20, in lean and obese Zucker rat liver. Dipeptidylpeptidase-IV (DPP-IV), crucial for the inactivation of incretins and neuropeptides, was used as a marker. Liver morphology and canalicular network of lean livers were similar after CS/reperfusion or MP20/reperfusion. CS preservation of fatty livers induced serious damage to the parenchyma and to the canalicular activity/expression of DPP-IV whereas with MP20 the morphology and canalicular network were similar to those of untreated lean liver. CS and MP20 had similar effects on DPP-IV activity and expression in the upper segments of the intrahepatic biliary tree of fatty livers. DPP-IV expression was significantly increased after MP20 respect to CS or to the controls, both for lean and obese animals. Our data support the superiority of MP20 over CS for preserving fatty livers. Dipeptidylpeptidase-IV activity and expression reveal decreased damage to the intrahepatic biliary tree in fatty livers submitted to subnormothermic machine-perfusion respect to conventional cold storage.

Dipeptidylpeptidase--IV, a key enzyme for the degradation of incretins and neuropeptides: activity and expression in the liver of lean and obese rats.

Given the scarcity of donors, moderately fatty livers (FLs) are currently being considered as possible grafts for orthotopic liver transplantation (OLT), notwithstanding their poor tolerance to conventional cold preservation. The behaviour of parenchymal and sinusoidal liver cells during transplantation is being studied worldwide. Much less attention has been paid to the biliary tree, although this is considered the Achille's heel even of normal liver transplantation. To evaluate the response of the biliary compartment of FLs to the various phases of OLT reliable markers are necessary. Previously we demonstrated that Alkaline Phosphatase was scarcely active in bile canaliculi of FLs and thus ruled it out as a marker. As an alternative, dipeptidylpeptidase-IV (DPP-IV), was investigated. This ecto-peptidase plays an important role in glucose metabolism, rapidly inactivating insulin secreting hormones (incretins) that are important regulators of glucose metabolism. DPP-IV inhibitors are indeed used to treat Type II diabetes. Neuropeptides regulating bile transport and composition are further important substrates of DPP-IV in the enterohepatic axis. DPP-IV activity was investigated with an azo-coupling method in the liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. Protein expression was studied by immunofluorescence with the monoclonal antibody (clone 5E8). In Wistar rat liver, DPP-IV activity and expression were high in the whole biliary tree, and moderate in sinusoid endothelial cells, in agreement with the literature. Main substrates of DPP-IV in hepatocytes and cholangiocytes could be incretins GLP-1 and GIP, and neuropeptides such as vasoactive intestinal peptide (VIP) and substance P, suggesting that these substances are inactivated or modified through the biliary route. In lean Zucker rat liver the enzyme reaction and protein expression patterns were similar to those of Wistar rat. In obese rat liver the patterns of DPP-IV activity and expression in hepatocytes reflected the morphological alterations induced by steatosis as lipid-rich hepatocytes had scarce activity, located either in deformed bile canaliculi or in the sinusoidal and lateral domains of the plasma membrane. These findings suggest that bile canaliculi in steatotic cells have an impaired capacity to inactivate incretins and neuropeptides. Incretin and/or neuropeptide deregulation is indeed thought to play important roles in obesity and insulin-resistance. No alteration in enzyme activity and expression was found in the upper segments of the biliary tree of obese respect to lean Zucker and Wistar rats. In conclusion, this research demonstrates that DPP-IV is a promising in situ marker of biliary functionality not only of normal but also of fatty rats. The approach, initially devised to investigate the behaviour of the liver during the various phases of transplantation, appears to have a much higher potentiality as it could be further exploited to investigate any pathological or stressful conditions involving the biliary tract (i.e., metabolic syndrome and cholestasis) and the response of the biliary tract to therapy and/or to surgery.

Response of renal parenchyma and interstitium of Rana snk. esculenta to environmental pollution.

The mesonephroi of two groups of Rana esculenta collected from two rice fields near Pavia, one relatively unpolluted and one polluted, were morphologically and histochemically investigated. Light and electron microscopy analyses were performed and certain enzyme activities studied (succinic dehydrogenase, SDH, alkaline phosphatase, AlkPase, acid phosphatase, AcPase, catalase, CAT, and NOS-related nicotinamide adenine dinucleotide phosphatase, NOS/NADPHd). The expression of the inducible NOS (iNOS) was evaluated through immunohistochemistry. In the renal parenchyma of the polluted group some structural modifications, mainly in the glomeruli and the proximal tubule epithelium, were observed. Peritubular inflammatory foci in most polluted samples were often found to be in combination with parasitic cysts. However, no necrotic processes were found in the renal parenchyma. Compared to controls, the histochemical studies on contaminated frogs evidenced an increase of the AcPase, NOS and CAT activities, and of the iNOS immunoexpression as well. All the results showed a good correspondence between the biomarkers responses and the environmental stress conditions. Overall, we can state that studying the sub-lethal effects of contamination in amphibians naturally exposed to toxicants has shown to be significant for the assessment of site-specific risk and potential hazards behind the phenomenon of progressive amphibian decline.

Decreased apoptosis in fatty livers submitted to subnormothermic machine-perfusion respect to cold storage.

Machine perfusion at subnormothermic temperature (20°C), MP20, was developed by Vairetti et al. and showed to afford a better preservation of fatty livers respect to traditional cold storage (CS) in terms of enzyme release into the perfusate and bile, glycogen stores, energy charge and oxidative stress. Here we investigated whether it also caused decreased cell death by apoptosis. Fatty and lean Zucker rats were submitted to MP20 or CS for 6 h and reperfused normothermically for 2 h. Apoptotic cells were revealed by immunohistochemistry of activated caspase-3 and M30 (new epitope on CK18 degraded by caspase-3) and by the TUNEL assay. Portal pressure was also determined. A statistically significant reduction of hepatocyte apoptosis, but especially of sinusoidal cells was determined for fatty livers submitted to MP20 respect to CS. Portal pressure was significantly lower after MP20 respect to CS. The reduction of sinusoidal cell death by apoptosis without need for anti-apoptotic therapies appears particularly positive since apoptotic sinusoidal cells hinder microcirculation in the sinusoids and are thrombogenic. These results further confirm the potential of MP20 for preserving fatty livers that would be otherwise discarded as grafts, and thus for increasing the donor pool for liver transplantation.

Morphofunctional evidence of changes in principal and mitochondria-rich cells in the epidermis of the frog Rana kl. esculenta living in a polluted habitat.

The epidermis of vertebrates is the body's principal barrier against environment and its possible contaminants. The presence of keratins, as well as specific detoxifying molecules or enzyme activities, in the various epidermis layers is believed to be involved in providing protection from harmful environmental influences. Anuran integument is poorly hornified and thus permeable to some endogenous and exogenous compounds and thus serves as a good bioindicator of overall environmental conditions. In the present investigation, we studied the epidermis of Rana kl. esculenta adult specimens collected at two different rice fields, relatively unpolluted and heavily polluted, respectively. Environmental pollution was assayed by chemical analysis performed on both sediments and animals. We evaluated the structural aspects of the epidermis at both light and electron microscopy levels and the pattern of keratinization by immunohistochemistry. Furthermore, we studied the activities of some enzymes (acid and alkaline phosphatase, nitric oxide synthase-related nicotinamide adenine dinucleotide phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, catalase, nonspecific esterases, and succinic dehydrogenase) involved mainly in membrane transport, xenobiotics, and oxidative metabolism. Compared with controls, in polluted animals we found the following results: (1) an increase in pollutant levels (i.e., cadmium, mercury, and lead); (2) less keratinized superficial cells in the epidermis; and (3) changes in most enzyme activities in keratinocytes and mitochondria-rich cells (particularly glucose-6-phosphate dehydrogenase and esterases, both important to counteract oxidative and toxic stress). Taken as a whole, the present data indicate the morphofunctional plasticity of the frog epidermis in response to environmental contamination.

Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery.

Herpes simplex virus thymidine kinase (HSV-TK) is widely used in gene therapy. The enzymatic activity of HSV-TK may be traced in vivo by specific radiopharmaceuticals in order to image transgene expression. However, most of these radiopharmaceuticals are toxic per se or after activation by HSV-TK, and therefore do not represent ideal molecules for clinical applications and repeated imaging. Unlike human cytosolic TK, HSV-TK is not enantioselective and can efficiently phosphorylate both D and L enantiomers of beta-thymidine. Here we show that, after phosphorylation by HSV-TK, tritiated L-beta-thymidine (LT) is selectively retained inside the cells in vitro and in vivo. We used the in vivo accumulation of radioactive phosphorylated LT to image the HSV-TK-positive cells inside a transplantable murine brain tumour after inoculation of cells producing retroviruses carrying HSV-TK. Owing to their unnatural enantiomeric conformation, phosphorylated LT metabolites are very poorly processed by mammalian enzymes, thus leading to increased cellular retention and minimal toxicity. The ability to image cells expressing the HSV-TK gene by using radiolabelled LT, without damaging the cells accumulating the phosphorylated L-nucleoside, will be important to monitor the levels and spatial distribution of therapeutic vectors carrying HSV-TK.

Shc signaling in differentiating neural progenitor cells.

Previously we found that the availability of ShcA adapter is maximal in neural stem cells but that it is absent in mature neurons. Here we report that ShcC, unlike ShcA, is not present in neural stem/progenitor cells, but is expressed after cessation of their division and becomes selectively enriched in mature neurons. Analyses of its activity in differentiating neural stem/progenitor cells revealed that ShcC positively affects their viability and neuronal maturation via recruitment of the PI3K-Akt-Bad pathway and persistent activation of the MAPK pathway. We suggest that the switch from ShcA to ShcC modifies the responsiveness of neural stem/progenitor cells to extracellular stimuli, generating proliferation (with ShcA) or survival/differentiation (with ShcC).

Rack1 binds HIV-1 Nef and can act as a Nef-protein kinase C adaptor.

Nef proteins of primate immunodeficiency viruses exert pleiotropic effects, such as enhanced endocytosis of CD4 and MHC-I cell surface molecules, perturbation of signal transduction cascades, and virion infectivity enhancement. Nef function intersects that of a number of cell kinases, including C kinases (PKCs) and Src-family kinases. Here the interaction of HIV-1 Nef with Rack1 (receptor for activated C kinase 1) is reported. Nef binds the Rack1 C-terminal moiety in a yeast two-hybrid system and in cell-free pull-down assays and copurifies with in vitro translated Rack1. Nef and Rack1 partially colocalize on the trans-Golgi network and plasma membranes. The presence of Rack1 doubles Nef phosphorylation by PKCs in vitro. Our data agree with the idea that Rack1 acts as a Nef intracellular docking site, bringing Nef and PKCs together. Other signal transduction or endocytosis proteins, in particular Src-like kinases, might meet Nef by intermediation of the Rack1 adaptor.

Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein.

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.

Vitamin D metabolites activate the sphingomyelin pathway and induce death of glioblastoma cells.

1 alpha, 25-dihydroxyvitamin D3 was previously shown to induce cell death in brain tumour cell lines when added to the medium at micromolar concentration. In this paper we show that Cholecalciferol, a poor ligand of the vitamin D receptor, also induces cell death of HU197 human glioblastoma cell line and early passages cultures derived from a recurrent human glioblastoma. This finding suggests that the effects of vitamin D metabolites on brain tumour cells are at least partially independent from the activation of the classic nuclear receptor pathway. Vitamin D metabolites have been shown to activate the sphingomyelin pathway inducing an increase in cellular ceramide concentration. We determined the levels of sphingomyelin ceramide and ganglioside GD3 in Hu197 cells after treatment with cholecalciferol. A significant increase in ceramide concentration and a proportional decrease in sphingomyelin was already present after 6 hours of cholecalciferol treatment when no morphological changes were visible in the cultures. Treatment with ceramides (N-acetylsphingosine or natural ceramide from bovine brain) of the same cells also induces cell death. Similarly, treatment of the same cells with bacterial Sphingomyelinase also results in cell death. The demonstration of an increase in intracellular ceramide after cholecalciferol treatment and the ability of ceramide to induce cell death suggest that the sphingomyelin pathway may be implicated in the effect of vitamin D metabolites on human glioblastoma cells. Inhibition of ceramide biosynthesis by fumonisin B1 treatment did not alter the dose response curve of HU197 cells to cholecalciferol. Insensitivity to fumonisin B1 together with a decrease in sphingomyelin content after cholecalciferol treatment indicate that activation of sphingomyelinase should be responsible for the increase in intracellular ceramide concentration.

Intracellular retention of duck hepatitis B virus large surface protein is independent of preS topology.

The mechanism of intracellular retention for the large surface protein (L) of duck hepatitis B virus (DHBV) was analyzed by examination of the transmembrane topologies and secretory properties of a collection of DHBV L mutants and compared with that of human hepatitis B virus (HBV) L. Our results demonstrate that, in contrast to its HBV counterpart, intracellular retention of DHBV L does not depend on the cytosolic disposition of its preS domain. L mutants with either cytosolic or lumenal preS were mostly retained in the absence of the small surface protein (S), whereas coexpression with S resulted in efficient secretion of both topological forms. Coexpression of the wild-type DHBV L with S resulted in efficient incorporation of L into secreted S + L particles, whereas HBV L was partially excluded from secreted particles under the same conditions. We propose that HBV provides L retention even in the presence of an excess of S, by exclusion of molecules with cytosolic preS domains from secreted particles at the stage of their assembly. DHBV lacks such a retention mechanism due to the absence of topological selection in particulate assembly.

HsN3 proteasomal subunit as a target for human immunodeficiency virus type 1 Nef protein.

HIV-1 Nef protein is important for pathogenicity, but its biochemical function remains obscure. To clarify its role, a yeast two-hybrid system (ths) screening was utilized to identify Nef cellular partners. Of 79 yeast clones harboring cDNAs for putative Nef binding proteins, 27 (34%) contained the coding region for HsN3 proteasomal subunit. HsN3 behaved as bona fide Nef partner in ths control crosses. Nef-HsN3 interaction was confirmed by in vitro binding experiments. In particular, recombinant Nef was able to capture the HsN3 subunit from a natural proteasome preparation. In Nef, the interacting region was mapped within aa 34-143, which span the structured portion of the protein, including the SH3-binding domain. In HsN3, Nef-binding portion was restricted to aa 73-249, and the tract 219-249-reminiscent of SH3 domain N-terminal 3/5ths-was shown to be essential, though not sufficient. Attempts to purify a Nef-HsN3 complex from transfected COS7 cells were unsuccessful. However, Nef was found to markedly downregulate intracellular levels of both a coexpressed HsN3 and the endogenous simian homologue. These results suggest that Nef, by binding to a subunit, might alter proteasome function in infected cells.

Indobufen versus warfarin in the secondary prevention of major vascular events in nonrheumatic atrial fibrillation. SIFA (Studio Italiano Fibrillazione Atriale) Investigators.

BACKGROUND AND PURPOSE: The results of a large prospective randomized trial have shown the efficacy of oral anticoagulation in the secondary prevention of major vascular events in patients with nonrheumatic atrial fibrillation (NRAF); less well established is the role of antiplatelet agents. The present study compared the effects of indobufen, a reversible inhibitor of platelet cyclooxygenase, with those of warfarin in this setting. METHODS: A total of 916 patients with NRAF and a recent (< or = 15 days) cerebral ischemic episode were admitted to this multicenter, randomized study, during which they were treated with either indobufen (100 or 200 mg BID) or warfarin (to obtain an international normalized ratio of 2.0 to 3.5) for 12 months. The two groups (462 on indobufen and 454 on warfarin) were well balanced in terms of their main baseline characteristics. The primary outcome of the study was the combined incidence of nonfatal stroke (including intracerebral bleeding), pulmonary or systemic embolism, nonfatal myocardial infarction, and vascular death. RESULTS: At the end of follow-up, the incidence of primary outcome events was 10.6% in the indobufen group (95% confidence interval, 7.7% to 13.5%) and 9.0% in the warfarin group (95% confidence interval, 6.3% to 11.8%), with no statistically significant difference between treatments. The frequency of noncerebral major bleeding complications was low: only four cases (0.9%) of gastrointestinal bleeding were observed, all of them in the warfarin group. CONCLUSIONS: We conclude that, within the limitations of its design, this study may help the medical community in devising appropriate antithrombotic strategies for NRAF patients for whom oral anticoagulants are contraindicated or do not represent a feasible approach to treatment.

Common localization of retention determinants in hepatitis B virus L protein from different strains.

Hepatitis B virus L protein is retained intracellularly, and trans-inhibits secretion of the related S and M proteins, as particulate HBsAg, at high L/S-M ratios. Comparison of equivalent A and D strain mutants suggested that the retention mechanism does not vary with genotype. Contrary to an earlier suggestion, the N-terminal extension specific for A-C strains was found to be inactive as a retention signal. Intact L was more completely retained than any mutated protein. Retained mutants had either a critical PreS stretch, or N-terminal myristate. Also, mutants of the latter class did not completely inhibit particulate budding, and could, in minor amounts, reach the Golgi. We conclude that (i) the principal retention determinant can be traced to the same PreS segment in distinct strains and (ii) myristic acid does reinforce retention in wild-type L, while acting in part as an HBsAg membrane anchor in mutants lacking the internal determinant.

Human cytomegalovirus pp65 lower matrix phosphoprotein harbours two transplantable nuclear localization signals.

Human cytomegalovirus phosphoprotein pp65 is targeted to the cell nucleus immediately after infection. Deletion and point mutation analysis of the pp65 gene expressed in insect cells showed that two hydrophilic regions (HP1 and HP2) within the pp65 C-terminal 40% each harboured an independent nuclear localization signal (NLS); strong association to the nuclear stroma also requires the N-terminal domain. Either region, when fused to chloramphenicol acetyltransferase, localized the reporter protein to the nucleus in insect cells as well as in NIH 3T3 cells and human lung fibroblasts. In addition, HP1 was found to be the target of pp65 Ser/Thr phosphorylation in insect cells and a prokaryotically expressed HP1 was actively phosphorylated in vitro by casein kinase II, for which two site clusters map in HP1. These findings indicate that pp65 includes two NLSs, one of which has the potential to be modulated by phosphorylation.

Nef-CD4 physical interaction sensed with the yeast two-hybrid system.

HIV-1 Nef protein has been known to induce downmodulation of CD4 receptor. In order to test whether the two proteins physically interact, the yeast two-hybrid system was exploited. A Saccharomyces cerevisiae strain carrying a GAL4-responsive lacZ fusion gene was cotransformed with plasmids in which the Nef and the CD4 cytoplasmic domain (CD4cd) coding sequences were fused to either the DNA binding (DB) or the activation (A) moiety of the GAL4 transcriptional activator. Both the DB-Nef + A-CD4cd and the DB-CD4cd + A-Nef combinations activated the reporter gene, weakly but specifically, as inferred by comparison with a number of controls. Reporter activation was similary observed when DB-Nef was cotransfected with the fusion A-CD4cd(aa 1-23). On the contrary, the combination DB-Nef + A-CD4cd(aa 24-40) was inactive. Also, mutating the CD4cd Leu20-Leu21 motif (known to be essential for both physiological and Nef-induced CD4 endocytosis) to Ala20-Ala21 abolished the GAL4 activity of DB-Nef + A-CD4cd. None of six DB-Nef derivatives in which Nef was partially deleted activated specifically the reporter when coexpressed with A-CD4cd. These findings suggest that CD4cd and Nef directly interact and that a largely complete Nef is required for the interaction. CD4cd aa 1-23 are sufficient for binding; in particular, the Leu20-Leu21 motif is essential. One can infer from these data that: (i) Nef-induced CD4 downmodulation involves a direct CD4-Nef contact and (ii) CD4cd Leu20-Leu21 is required in Nef-induced downmodulation, not simply as an endocytosis signal, but also as an essential component of the Nef-binding moiety.

Characterization of human DNA ligase I expressed in a baculovirus-insect cell system.

The baculovirus expression system was used to overexpress human DNA ligase I (hLig I). Approx. 2 mg of recombinant hLig I was produced per 10(8) Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus. The optimum time point for the production of biologically active recombinant hLig I was 48 h post-infection. Lig I activity was demonstrated by auto-adenylating, polynucleotide joining and DNA relaxation assays. The baculovirus system has the advantage over previously described methods for producing hLig I of generating large amounts of a full-length active protein.

A C-terminal PreS1 sequence is sufficient to retain hepatitis B virus L protein in 293 cells.

Analysis of deletion and/or site-specific mutants of the hepatitis B virus (HBV) env gene, expressed in human cells, provided clues about the mechanism that retains the L protein, the largest gene product, in a pre-Golgi compartment. Differences in secretability of the analyzed variants suggest that the N-terminal myristic acid and an internal sequence within the PreS1 region function as independent retention signals. N-terminal myristic acid alone neither prevented PreS1 + 2 N-linked glycosylation, which signals cotranslational translocation of the domain, nor strongly inhibited lumenal budding. Thus, myristic acid by itself acts by arresting secretion of lumenal, soluble Env particles. By contrast, the internal retention determinant, mapping in the C-terminal portion of PreS1, also prevented budding. In addition, the presence of this PreS1 segment correlated with the depression of PreS1 + 2 glycosylation. This suggests a connection between L retention and the recently described inhibition of PreS1 + 2 cotranslational translocation. A model can be proposed, according to which HBV surface proteins need to cotranslationally translocate their N-terminal moieties in order to assume a transmembrane topology suitable for particulate assembly and secretion. L protein, whose PreS1 + 2 domain undergoes translocation only posttranslationally, would fail to complete the secretion process. To support this model, we show that forced cotranslational translocation of the PreS1 + 2 domain (by attachment of an N-terminal processed signal sequence) results in secretion of L protein.

Effects of vitamin D and retinoic acid on human glioblastoma cell lines.

The biological significance of vitamin D receptors expressed by glioblastoma and other glial tumours is still unclear. In an effort to clarify this issue we studied the effects of increasing concentrations of 25-dihydroxyvitamin D3 and its metabolite 1 alpha,25-dihydroxyvitamin D3 on two human glioblastoma cell lines. Both substances were capable of inducing a significant (> 50%) reduction in growth of the two glioblastoma cell lines at dosages over 5 microM. When the HU 70 cell line was treated by increasing dilutions of 25-dihydroxyvitamin D3 combined with 1 microM all trans-retinoic acid, significant inhibition was apparent even after addition of 25-dihydroxyvitamin D3 in the nanomolar range. Reduction of growth index was mainly due to induced cell death. Our results provide in vitro evidence that vitamin D metabolites alone or in combination with retinoids may be potentially useful agents in the differentiation therapy of human malignant gliomas.