Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene.

In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon-intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows: NH2 - lipovitellin I - phosvitin (or phosvette II - phosvette I) - lipovitellin II - COOH.

Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

Growth Arrest-Specific Gene 6 product modulates innate immunity in sepsis

Background: Growth Arrest-Specific Gene 6 product (Gas6) is, like anticoagulant protein C, a vitamin K-dependent protein. Our aim was to determine whether Gas6 plays a role in sepsis. Materials and methods: We submitted mice lacking Gas6 (Gas6)/)) or one of its receptors (Axl)/), Tyro3)/) or Mertk)/)) to LPS-induced endotoxemia and peritonitis (cecal ligation and puncture (CLP) and inoculation of E. coli). In addition, we measured Gas6 or its soluble receptors in plasma of eight volunteers that received LPS, 13 healthy subjects, 28 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Results: Gas6 and its soluble receptor sAxl raised in mice models and TNF-a was more elevated in Gas6)/) mice than in wild-type (WT). Protein array showed that before and after LPS injection, titers of 62 cytokines were more elevated in plasma of Gas6)/) than WT mice. Endotoxemia-induced mortality was higher in Gas6)/), Axl)/), Tyro3)/) and Mertk)/) compared to WT mice and mortality subsequent to CLP was amplified in Gas6)/) mice. LPS-stimulated Gas6)/) macrophages produced more cytokines than WT macrophages. This production was dampened by recombinant Gas6. Phosphorylation of Akt in Gas6)/) macrophages was reduced, but p38 phosphorylation and NF-jB translocation were increased. In human, Gas6 raised in plasma after LPS (2 ng/kg). Gas6 and sAxl were higher in patients with severe sepsis than in healthy subjects or control patients, and there was a non-significant trend for higher Gas6 in the survival group. Conclusions: Our data point to Gas6 as a major modulator of innate immunity and provide thereby novel insights into the mechanism of sepsis. Thus Gas6 and its receptors might constitute potential therapeutic targets for the development of new immunomodulating drugs.

Vaccinia immune globulin: current policies, preparedness, and product safety and efficacy.

In 1980 the World Health Organization declared that smallpox was eradicated from the world, and routine smallpox vaccination was discontinued. Nevertheless, samples of the smallpox virus (variola virus) were retained for research purposes, not least because of fears that terrorist groups or rogue states might also have kept samples in order to develop a bioweapon. Variola virus represents an effective bioweapon because it is associated with high morbidity and mortality and is highly contagious. Since September 11, 2001, countries around the world have begun to develop policies and preparedness programs to deal with a bioterror attack, including stockpiling of smallpox vaccine. Smallpox vaccine itself may be associated with a number of serious adverse events, which can often be managed with vaccinia immune globulin (VIG). VIG may also be needed as prophylaxis in patients for whom pre-exposure smallpox vaccine is contraindicated (such as those with eczema or pregnant women), although it is currently not licensed in these cases. Two intravenous formulations of VIG (VIGIV Cangene and VIGIV Dynport) have been licensed by the FDA for the management of patients with progressive vaccinia, eczema vaccinatum, severe generalized vaccinia, and extensive body surface involvement or periocular implantation following inadvertent inoculation.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.

SEPT4, a p53 target gene product, implicates p 53 in the regulation of exocytosis

Résumé : Le cancer, qui est responsable d'un quart des décès en Suisse, exhibe un état cellulaire désordonné, qui lui-même, est la conséquence d'un dérèglement des gènes. Le gène le plus fréquemment altéré, dans les cas de cancers humains, est p53. Ce gène encode un facteur de transcription, impliqué dans la régulation de nombreux gènes impliqués dans le cycle cellulaire, l'apoptose ou la différenciation. Notre laboratoire a récemment identifié seize nouveaux gènes, dont l'expression est régulée par p53, parmi lesquels sept4, su jet de cette thèse. La protéine 5EPT4 appartient à la famille des septines, qui est impliquée dans la cytokinèse. Dans ce travail, nous avons confirmé la régulation de l'expression de sept4 par p53 dans des tissus de souris, et étonnamment, seul un des deux promoteurs du gène sept4 est contrôlé par p53. En outre, l'approche immunohistologique nous a permis de supposer une implication de la protéine SEPT4 dans le mécanisme de l'exocytose. Cette hypothèse a été confirmée par l'interaction de SEPT4 avec la protéine syntaxine 1A, et par son activité inhibitrice sur la sécrétion stimulée. En élargissant l'étude de la protéine SEPT4, nous avons découvert que celle-ci avait comme partenaire fonctionnel, la protéine Pinl, une enzyme qui catalyse l'isomérisation cis-trans du lien peptidique précédant une proline. bans ce contexte, nous avons démontré que l'interaction entre ces deux protéines reposait sur le domaine WW de Pinl, un type de domaine reconnaissant les motifs phosphoséryl-prolyl et phosphothréonyl-prolyl. Ce dernier résultat nous a conduit à examiner la phosphorylation de 5EPT4. Nous avons démontré que la partie N-terminale de SEPT4 était phosphorylée par la kinase Cdk5. La co¬expression de Cdk5 et de SEPT4 stimule la dégradation de SEPT4, indépendamment de la voie du protéasome. Ainsi, l'ensemble de nos observations fournissent l'évidence de l'engagement de la protéine SEPT4 dans la régulation de l'exocytose, et soutiennent le rôle de p53 dans le contrôle de l'exocytose, via SEPT4, ce qui constituerait un nouveau rôle fonctionnel pour ce gardien du génome. Summary: Cancer, which is responsible for a quarter of the deaths in Switzerland, exhibits a disordered cellular state, which itself, is the consequence of an altered state of genes. The most frequently altered gene in human cancer is p53. This gene encodes a transcription factor, implicated in the regulation of numerous genes involved in cell cycle, apoptosis or differentiation. Our laboratory has recently identified sixteen new genes whose expression is regulated by p53, amongst them septin 4, which is the subject of this thesis. The SEPT4 protein belongs to the septin family which is implicated in cytokinesis. In the present work, we have confirmed the regulation of sept4 expression by p53 in mouse tissues, and surprisingly, only one of the two sept4 promoters is regulated by p53. In addition, the immunohistologic approach enabled us to suppose a role of SEPT4 in exocytosis. This assumption was confirmed by the interaction of SEPT4 with syntaxin 1A, and by its inhibiting activity on stimulated secretion. By widening the analysis of SEPT4, we identified Pin1 as an interacting protein. Pin1 is an enzyme which catalyzes the cis-trans isomerization of the peptide bond preceding a proline residue. In this context, we showed that the interaction between these two proteins depends on the WW domain of Pin 1. This domain has been shown to function as a phosphoserine- or phosphothreonine¬binding module. This last result prompted us to examine phosphorylation of SEPT4. We demonstrated that the N-terminal portion of SEPT4 was phosphorylated by the Cdk5 kinase. The co-expression of Cdk5 with 5EPT4 stimulates SEPT4 degradation, independently of the proteasome pathway. Thus, these observations provide evidence for the engagement of SEPT4 in the regulation of exocytosis, and supports the role of p53 in the control of exocytosis, via SEPT4, which constitutes a new functional role for this guardian of the genome.

French summaries of product characteristics: content in relation to therapeutic monitoring of psychotropic drugs.

The prescription information (summary of product characteristics, SPC) is compiled by the pharmaceutical industry as required by the national regulatory authorities. They vary in their content about the properties of drugs and about the usefulness of therapeutic drug monitoring (TDM) in the blood of patients. Based on a previous study carried out in Germany, the degree of agreement of French SPC for 59 psychotropic drugs with the existing medico-scientific evidence in the area of TDM was examined using a recently developed instrument. A summary score of SPC content (SPCC) related to TDM (SPCC(TDM)) has been calculated and compared with the level of recommendation of TDM of the AGNP-TDM expert group consensus guidelines for TDM in psychiatry [AGNP: Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (Association for neuropsychopharmacology and pharmacopsychiatry)]. Among the antidepressants, antipsychotics, tranquillizers/hypnotic agents and mood stabilizers, the highest SPCC(TDM) scores in the French SPC were reached for imipramine (16), haloperidol (6), clonazepam (8) and lithium (23), respectively. Results were similar to those obtained from the analysis of German SPC, and considerable disagreement was found between the information on TDM in SPC and existing medico-scientific evidence, albeit less in the case of mood stabilizers. Taking into account the recommendations of the AGNP-TDM expert group guidelines, there is a deficit in the French SPC concerning TDM-relevant information. An amelioration of this situation could help to improve the clinical practice of TDM of psychotropic drugs, as the SPC is a widely used tool.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.