The Case of Pharmacological Neuroenhancement: Medical, Judicial and Ethical Aspects from a German Perspective.

Pharmacological neuroenhancement (PN) describes the use of psychoactive drugs for the purpose of enhancing cognition (e. g., fatigue, concentration, memory etc.) by healthy subjects without medical need. Drugs used for this purpose can be divided into freely available, over-the-counter drugs (e. g., methylxanthines such as caffeine), prescription drugs (e. g., antidementia drugs, methylphenidate) and illicit drugs (e. g., illicit amphetamines). Clinical studies have shown that the aforementioned substances only have limited pro-cognitive effects and have considerable safety risks and side effects.The German judicial perspective shows legal differences between substances (drugs, food, food supplements, fortified food) that can be bought in a supermarket, drugs that can be bought in a pharmacy as over-the-counter- (OTC-) drugs, drugs with or without the need for a prescription and illicit drugs. Supermarket drugs and fortified food can be sold freely and follow the general rules of civil and penal law; regarding acquisition, parents are responsible for their children. OTC drugs require special information about therapy. Regarding prescription drugs, there are legal problems caused by an off-label use and the non-medical purposes of PN drugs. Furthermore, prescription stimulants for PN are governed by the specialized law for narcotics, and their use might be punished. Beyond the general lack of rules for regulation for PN drug use there are specific needs for prevention (e. g., control of the black market, etc.).Possible future policy will depend, among others, on the probability with which effective PN drugs with an acceptable risk-benefit ratio will be available, on individual and societal implications, and on public opinion towards PN. While 4 different general policy scenarios can be identified, it is important to advance a broad societal debate on PN to collect relevant empirical data and to address enhancement-related conceptual issues.

Non-medical use of prescription stimulants and illicit use of stimulants for cognitive enhancement in pupils and students in Germany.

INTRODUCTION: The aim of this study was to assess for the first time the prevalence and factors associated with stimulant use exclusively for cognitive enhancement among pupils and university students in Germany. METHODS: A sample of 1 035 pupils (vocational and grammar schools) in small and big cities and 512 university students of 3 Departments (Medicine, Pharmacy, Economics) completed a questionnaire regarding knowledge and use of stimulants for cognitive enhancement and factors associated with their use. RESULTS: Lifetime prevalence for use of prescription stimulants (methylphenidate, amphetamines) for cognitive enhancement in pupils was 1.55% and in students 0.78%. Last-year and last-month prevalence rates were significantly lower. 2.42% of pupils and 2.93% of students reported lifetime illicit use of stimulants (amphetamines, cocaine, ecstasy) for cognitive enhancement with lower last-year and last-month rates. Prevalence was higher in male pupils, pupils from vocational schools and pupils with bad marks. DISCUSSION: The illicit use of stimulants for cognitive enhancement is significantly higher than non-medical use of prescription stimulants among pupils and students. Stimulant use is determined by gender, school type, and school marks. The potential risks associated with stimulant use require early awareness and intervention strategies.

Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine. Its pleiotropic biological properties are signaled through two distinct cell surface receptors: the TNF receptor type I (TNFR-I) and the TNF receptor type II. Neither of the two receptors possesses tyrosine kinase activity. A large majority of TNF-alpha-dependent activities can be mediated by TNFR-I. Recently, c-Raf-1 kinase was identified as an intracellular target of a signal transduction cascade initiated by binding of TNF-alpha to TNFR-I. However, the mechanism engaged in TNF-alpha-dependent activation of c-Raf-1 kinase is still enigmatic. Here we report that the cytosolic adapter protein Grb2 is a novel binding partner of TNFR-I. Grb2 binds with its COOH-terminal SH3 domain to a PLAP motif within TNFR-I and with its NH2-terminal SH3 domain to SOS (son of sevenless). A PLAP deletion mutant of TNFR-I fails to bind Grb2. The TNFR-I/Grb2 interaction is essential for the TNF-alpha-dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF-alpha can be blocked by coexpression of Grb2 mutants harboring inactivating point mutations in the NH2- or COOH-terminal SH3 domain, cell-permeable peptides that disrupt the Grb2/TNFR-I interaction or transdominant negative Ras. Functionality of the TNFR-I/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF-alpha-dependent activation of c-Raf-1 kinase. Inhibition of the TNFR-I/FAN (factor associated with neutral sphingomyelinase) interaction, which is essential for TNF-alpha-dependent activation of the neutral sphingomyelinase, either by cell-permeable peptides or by deletion of the FAN binding domain, prevents activation of c-Raf-1 kinase. In conclusion, binding of the Grb2 adapter protein via its COOH-terminal SH3 domain to the nontyrosine kinase receptor TNFR-I results in activation of a signaling cascade known so far to be initiated, in the case of the tyrosine kinase receptors, by binding of the SH2 domain of Grb2 to phosphotyrosine.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. METHODS: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. RESULTS: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. CONCLUSION: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.

Impact of HBV genotype and mutations on HBV DNA and qHBsAg levels in patients with HBeAg-negative chronic HBV infection.

BACKGROUND: HBV DNA and quantitative (q)HBsAg levels as prognostic markers for HBV-related disease are mostly validated in Asia and their significance in Western populations is uncertain. AIM: To analyse the impact of the HBV genotype and frequent mutations in precore (PC), basal core promoter (BCP) and preS on HBV DNA and qHBsAg levels. METHODS: HBV DNA and qHBsAg serum levels of 465 patients with HBeAg-negative chronic HBV infection were correlated with the HBV genotype and mutations in PC, BCP and preS. For a detailed analysis of the molecular virology, genotype A2 genomes harbouring these mutations were analysed for replication efficacy and HBsAg release in cell culture. RESULTS: While no impact of the HBV genotype on HBV DNA levels was observed, qHBsAg levels differed up to 1.4 log among the genotypes (P < 0.001), reflected by large differences regarding the 1000 IU/mL HBsAg cut-off. While PC mutations were associated with higher (P < 0.001), BCP mutations were associated with lower HBV DNA levels (P < 0.001). Higher qHBsAg levels were associated with preS and lower levels with PC mutations (P < 0.001 and P = 0.001, respectively). The cell culture experiments revealed a higher HBsAg release and shorter filaments in case of a HBV genome harbouring a preS deletion. In contrast, a perinuclear HBsAg accumulation was detected for the PC and BCP-variants, reflecting an impaired HBsAg release. CONCLUSIONS: qHBsAg serum levels depend on the HBV genotype and together with HBV DNA levels on frequent mutations in PC, BCP and preS in HBeAg-negative patients. qHBsAg cut-offs when used as prognostic markers require genotype-dependent validation.

Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication.

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) is essential for viral replication. Here, the kinase Raf-1 was identified as a novel cellular binding partner of NS5A, binding to the C-terminal domain of NS5A. Raf-1 colocalizes with NS5A in the HCV replication complex. The interaction of NS5A with Raf-1 results in increased Raf-1 phosphorylation at serine 338. Integrity of Raf-1 is crucial for HCV replication: inhibition of Raf-1 by the small-molecule inhibitor BAY43-9006 or downregulation of Raf-1 by siRNA attenuates viral replication.

Characterization of essential domains for the functionality of the MHBst transcriptional activator and identification of a minimal MHBst activator.

Integrated hepatitis B virus DNA derived from hepatocellular carcinomas can express, in one third of the cases investigated so far, a transcriptional activator encoded from 3' terminal truncated surface (preS/S) genes resulting in a C-terminally truncated middle surface protein (MHBst). Since MHBst, in contrast to the secreted MHBs, is retained in the secretory pathway at the ER, the question as to whether the retention generates the transcriptional activator function was investigated. Through fusion of MHBs to the ER-retention signal KDEL, it was shown that the intracellular retention does not generate the transcriptional activator function. Tryptic digestions of microsomal vesicles revealed that the amino terminal domain of MHBst directs into the cytoplasmic compartment, whereas in MHBs this domain directs into the lumen of the ER. This structural difference appears to be why transcriptional activator function arises. Through deletion analysis it was shown that non-membrane-associated MHBst proteins are also functional activators. Nonmembrane associated MHBst proteins represent a second class of MHBst proteins. These MHBst-proteins are homogenously distributed all over the cell and show no difference in functionality as compared to the membrane-associated MHBst proteins. MHBst53 (truncated at aa53) was shown to be a minimal activator of this class. Both classes of MHBst proteins were found to form dimers; an which is involved in mediating the dimerization. The integrity of this domain was also revealed to be a prerequisite for the functionality of the activator, suggesting a linkage between dimerization and functionality.

ER-localization and functional expression of the HBV transactivator MHBst.

In two recently reported cases, integrated hepatitis B virus (HBV) DNAs cloned from hepatocellular carcinoma were found to express a transcriptional transactivator from 3'-terminally truncated HBV surface (preS/S) genes. In this study, we characterized the transactivator at the protein level. Expression of a 3'-truncated preS2/S gene in Spodoptera frugiperda (Sf9) insect cells resulted in a C-terminally truncated middle surface protein of 76 amino acids (MHBst76), which was found to be associated with membranes of the endoplasmic reticulum and retained from Golgi processing and secretion. Accordingly, the microsome fraction of MHBst76-expressing Sf9 cells displayed transactivator activity after electric field-mediated transfer into Chang liver cells. In contrast to full-length MHBs, MHBst76 is unglycosylated, and glycosylation is not required for transactivation as shown by mutation of the glycosylation site at asparagine-4. Since highly purified MHBst76 derived from an E. coli expression system also showed transactivator activity, it is concluded that unglycosylated MHBst76 protein is the authentic transactivating factor. As the transactivator protein derives from inactive MHbs by rearrangements of integrated HBV DNA, it may be important for HBV-associated liver carcinogenesis.

CD30-mediated cell cycle arrest associated with induced expression of p21(CIP1/WAF1) in the anaplastic large cell lymphoma cell line Karpas 299.

One of the major characteristics of anaplastic large cell lymphomas (ALCL) is the expression of the Ki-1/CD30 antigen. While the receptor mediates NF-kappaB-activation in Hodgkin's lymphomas, some data suggest the CD30-mediated apoptosis of other CD30-expressing cells. We were able to demonstrate that activation of CD30 leads to different effects regarding cell proliferation of the ALCL-derived cell lines Karpas 299 and JB6. Western and Northern blotting analysis revealed that CD30-induced growth inhibition of Karpas 299 cells correlated with a strong upregulation of the cell cycle inhibitor p21(CIP1/WAF1). We found a non activating point mutation at codon 273 in exon 8 of the p53 gene in Karpas 299 cells which indicates an p53-independent mechanism for induced p21 expression. Abundant p21 protein expression resulted in hypophosphorylation of the retinoblastoma protein (Rb) and inhibition of the proliferating cell nuclear antigen (PCNA). CD30-stimulated cells showed no indications of apoptotic cell death, like genomic DNA fragmentation or cleavage of the caspase-3 target protein poly (ADP-ribose) polymerase (PARP). Our results indicate that CD30 is able to mediate an p21-associated cell cycle arrest in ALCL with possible implications for prognosis and clinical treatment.

Anti-hepatitis B virus activity of N-acetyl-L-cysteine (NAC): new aspects of a well-established drug.

N-acetyl-L-cysteine (NAC) is commonly administered as an antidote against acetaminophen intoxication and is the preferred agent in the treatment of pulmonary diseases. It is furthermore commonly considered that it restrains human immunodeficiency virus (HIV) replication by scavenging reactive oxygen intermediates (ROI) and thus suppressing activation of nuclear factor kappa B (NF kappa B). We show here that NAC is in addition able to inhibit hepatitis B virus (HBV) replication, but by a mechanism independent of the intracellular level of reactive oxygen intermediates. Treatment of HBV-producing cell lines with NAC resulted in an at least 50-fold reduction of viral DNA in the tissue culture supernatant within 48 h. This decrease of viral DNA and thus of virions in the tissue culture supernatant is caused by a disturbance of the virus assembly, rather than by a reduction of viral transcripts. Our data strongly suggest a potential use of this well-established, non-toxic drug for the treatment of HBV infection. Since NAC, in contrast to interferon, exerts its anti-HBV activity at a posttranscriptional level, a combination of NAC with the established interferon therapy could also be considered.

The PreS2 activators of the hepatitis B virus: activators of tumour promoter pathways.

In addition to causing acute and chronic hepatitis, hepatitis B virus (HBV) is considered to be a major cliological factor in the development of human hepatocellular carcinoma (HCC). Epidemiological studies have demonstrated an approximately 10-fold increase in the relative risk of HCC among HBV carried compared to noncarriers. Almost all HBV-associated HCCs studied so far harbor chromosomally integrated HBV DNA. Integrated viral DNA can encode two types of transcriptional activators, the HBx protein and the PreS2 activators [the large surface proteins (LHBs) and truncated middle surface proteins (MHBs)]. The activator function of the PreS2 activators is based on the cytoplasmic orientation of the PreS2 domain. The PreS2 domain is PKC-dependent phosphorylated. Moreover, the PreS2 domain binds of PKC alpha/beta and triggers a PKC-dependent activation of the c-Raf-1/MAP2-kinase signal transduction cascade, resulting in an activation of transcription factors such as AP-1 and NF-kB. Furthermore, by activation of this signaling cascade, the PreS2 activators cause an increased proliferation rate of hepatocytes. According to the two-step model of carcinogenesis (initiation/promotion), the PreS2 activators could exert a tumour-promoter-like function by activation of the PKC/c-Raf-1/MAP2-kinase signaling cascade: cells harboring critical mutations (initiation) may be positively selected (promotion). Such a multistep process may account for the long latency period in HCC development, but it also leads to the hypothesis that each tumor reflects an individual case.

The translocation motif of hepatitis B virus improves protein vaccination.

Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses.

Ethical aspects of neural tissue transplantation.

The method of neural grafting is considered to be a very promising therapeutic strategy for the treatment of certain neurodegenerative disorders such as Parkinson's disease or Huntington's disease. During the last 15 years, clinical transplantation studies have been carried out worldwide in several hundreds of patients with Parkinson's disease. In these studies, primarily fetal mesencephalic tissue derived from aborted human fetuses has been used for implantation. Neural tissue transplantation gives rise to ethical issues in two different areas that need careful examination: the first, ethical problems linked to the use of tissue from aborted human fetuses; and the second, ethical issues concerning the graft recipients in clinical trials, i.e., his or her well-being, personality, and personal identity.

Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens.

Efficient transfer of proteins or nucleic acids across cellular membranes is a major problem in cell biology. Recently the existence of a fusogenic sequence was predicted in the junction area of the PreS2- and S-domain of the hepatitis-B virus surface antigens. We have identified cell permeability as a novel property of the PreS2-domain. Cell permeability of PreS2 is not restricted to hepatocytes. PreS2 translocates in an energy-independent manner into cells and is evenly distributed over the cytosol. Detailed analysis revealed that cell-permeability is mediated by an amphipatic alpha-helix between amino acids 41 and 52 of PreS2. Destruction of this translocation motif (PreS2-TLM) abolishes cell permeability. PreS2-TLM per se can act as a shuttle for peptides and functional proteins (such as EGFP). This permits the highly specific modulation of intracellular signal transduction by transfer of peptides competing protein-protein interactions as demonstrated by specific inhibition of TNFalpha-dependent activation of c-Raf-1 kinase. Moreover in vivo functionality was demonstrated by PreS2-TLM-dependent protein transfer into primary bone marrow cells and into the liver. The amphipatic motif is conserved between the different hepatitis-B virus subtypes, and the surface proteins of avian and rodent hepadnaviruses exhibit similar amphipatic peptide sequences. In respect to hepatitis-B virus-infection, the PreS2-TLM could represent the postulated fusion peptide and play a crucial role in the internalization of the viral particle.

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Isolation of highly purified, functional carboxy-terminally truncated hepatitis B virus middle surface protein activators from eucaryotic expression systems.

Carboxy-terminally truncated hepatitis B virus (HBV) middle surface proteins (MHBst) show a transcriptional activator function. Two different subtypes of MHBst activators can be distinguished: an ER-localized type, represented here by MHBst76 (truncated at amino acid 76), and a cytosol-localized type, represented here by MHBst63. To characterize the MHBst activator on the protein level and to analyze posttranslational modifications, we established recombinant baculoviruses encoding for fusion proteins of MHBst76 or MHBst63 and of an amino terminal hexa-his tag. Both proteins could be obtained in high purity by affinity chromatography using Ni-nitrilo-tri-acetate agarose. In addition, 6H-MHBst76 was also isolated from transiently transfected HepG2 cells. Both the Spodoptera frugiperda (Sf9) cell-derived and the HepG2 cell-derived MHBst proteins were found to be unglycosylated. A detailed analysis of Sf9 cell-derived 6H-MHFBst76 by electrospray-ionization mass spectrometry showed that a fraction of this protein is N-terminally acetylated and phosphorylated or sulfated. Electric-field-mediated transfer of the highly purified proteins into reporter cells demonstrated that the isolated proteins are functional transcriptional activators. These experiments further showed that Sf9 cell-derived and HepG2 cell-derived 6H-MHBst do not differ in their functionality. This system allowed production and purification of functional 6H-MHBst in amounts sufficient enough to allow a further detailed analysis of MHBst activators on the protein level.

The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

Isolation and molecular characterization of hepatitis B virus X-protein from a baculovirus expression system.

The X protein (HBx) of the human Hepatitis B Virus (HBV) is a regulatory protein that exercises a transcriptional activator function on a variety of regulatory elements and is therefore considered to be involved in the development of human hepatocellular carcinoma (HCC). So far, most attempts at elucidating HBx function have been undertaken at the genetic level, reflecting the difficulties in detecting the very low amounts of the protein in infected livers. Consequently, the questions of intracellular localization and posttranslational modification have not yet been completely answered. We therefore constructed recombinant baculoviruses that allowed expression of HBx and the hexa histidine HBx fusion protein HBxHis in insect cells. Cell fractionation experiments revealed that only a minor part of HBx is detectable in a soluble form in the cytosolic fraction, whereas most of the protein forms intracellular aggregates. These results could be confirmed by confocal laser immunofluorescence. The fusion of a hexa-histidine tag to the amino terminus of HBx allowed a rapid one-step purification by metal chelate affinity chromatography. The detailed analysis of purified HBxHis using electrospray ionization mass spectrometry uncovered two major components: the unmodified, monomeric, fully oxidized form with five intramolecular disulfide bridges, and its N-acetylated modification. Additionally, two minor peaks with mass differences of delta m = +80 da suggested that a small fraction of HBx becomes posttranslationally phosphorylated in insect cells. No further modifications could be observed, indicating that only phosphorylation might play a role in a possible posttranslational regulation of this viral activator.