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Patients with Behçet's disease (BD) constantly complain of fatigue and many have problems with poor sleep. This ultimately has a major impact on all aspects of normal living. To attempt to understand this, Artificial Intelligence (AI) was used to identify potential biomarkers. These were alpha-melanocyte stimulating hormone (α-MSH), vasoactive intestinal peptide (VIP) and some inflammatory cytokines. We assessed the association of fatigue, quality of sleep and disease activity with circulating concentration of α-MSH, VIP and inflammatory cytokines.There were 127 participants, 97 BD patients, and 30 healthy controls (HC). All completed the Multi-Dimensional Assessment of Fatigue questionnaire (MAF) and the Pittsburgh Sleep Quality Index (PSQI) on the day of their clinical assessment. Enzyme-linked immunosorbent assays (ELISA) were used to evaluate the serum concentrations of α-MSH, VIP and cytokines (IL-1β, IL-6, IL-10, and TNF-α).64% of BD patients experienced high fatigue scores, and 63% had poor quality of sleep. When BD and HC were compared the MAF and PSQI scores as well as the serum concentrations of α-MSH, VIP, and IL-6 were significantly higher in BD (p values were: 0.001, 0.001, 0.001, 0.004 and 0.036, respectively). Both α-MSH and IL-6 had significant impact on MAF and PSQI. Interestingly, VIP had a significant influence on PSQI and disease activity, but not on MAF.A better understanding of these complex clinical and biochemical interactions between α-MSH, VIP and IL-6 might lead to the development of novel approaches to manage fatigue and sleep disorders as well as disease activity in BD patients.

Rationale: A deficiency of the pulmonary vasodilative vasoactive intestinal peptide (VIP) has been suggested to be involved in the pathophysiology of pulmonary hypertension (PH). Supplementation of VIP as an aerosol is hampered by the fact that it is rapidly inactivated by neutral endopeptidases (NEP) located on the lung surface. Hypothesis: Co-application of thiorphan, a NEP 24.11 inhibitor, could augment the biological effects of inhaled VIP alone. Methods: A stable pulmonary vasoconstriction with a 3-fold increase of pulmonary artery pressure was established by application the thromboxane mimetic U46619 in the isolated rabbit lung model. VIP and thiorphan were either applied i.v. or as an aerosol. Results: VIP caused a significant pulmonary vasodilation either during i.v. application or inhalation. These effects were of short duration. Thiorphan application had no effects on pulmonary vasoconstriction per se, but significantly augmented the effects of VIP aerosol. Thiorphan not only augmented the maximum hemodynamic effects of VIP aerosol, but also led to a significant prolongation of these effects. Conclusion: VIP causes pulmonary vasodilation in a model of acute experimental PH. The hemodynamic effects of VIP aerosol can be significantly augmented via co-application of a NEP inhibitor.

A novel series of amidinohydrazone-derived furin inhibitors was prepared; the most potent compounds 17 and 21 inhibit furin with Ki values of 0.46 and 0.59 μM, respectively. In contrast to inhibitor 17, which still contains a guanidino residue, compound 21 possesses only weakly basic amidinohydrazone groups.

Rationale: Previous studies suggest an important immunoregulatory role of vasoactive intestinal peptide (VIP) in experimental models of chronic noninfectious inflammation. Sarcoidosis is characterized by noncaseating epitheloid cell granulomas, where excessive tumor necrosis factor-α production by pulmonary macrophages plays a critical role in granuloma formation and disease progression, which may lead to fatal organ dysfunction. Objectives: To test whether inhaled VIP has an immunoregulatory role. Sarcoid alveolitis was used as a prototype of immune-mediated chronic lung inflammation. Methods: In an open clinical phase II study, we treated 20 patients with histologically proved sarcoidosis and active disease with nebulized VIP for 4 weeks. Measurements and Main Results: VIP inhalation was safe, well-tolerated, and significantly reduced the production of tumor necrosis factor-α by cells isolated from bronchoalveolar lavage fluids of these patients. VIP treatment significantly increased the numbers of bronchoalveolar lavage CD4 +CD127-CD25+ T cells, which showed regulatory activities on conventional effector T cells. In vitro experiments demonstrated the capacity of VIP to convert naive CD4+CD25- T cells into CD4+CD25+FoxP3+ regulatory T cells, suggesting the generation of peripheral regulatory T cells by VIP treatment. Conclusions: This study is the first to show the immunoregulatory effect of VIP in humans, and supports the notion of inhaled VIP as an attractive future therapy to dampen exaggerated immune responses in lung disorders. Thus, the inhalation of neuropeptides may be developed into a new therapeutic principle for chronic inflammatory lung disorders in humans.

Introduction Inhalation of vasoactive substances is an effective treatment of pulmonary hypertension. The B-type natriuretic peptide (BNP) leads to relaxation of smooth muscle cells, caused by an increased formation of cyclic guanosine monophosphate (cGMP). The biologic activity of BNP using an inhalative approach has not been addressed. Methods In order to assess the vasorelaxing capacity of exogenous BNP in the isolated ventilated and buffer perfused rabbit lung model, a stable pulmonary vasoconstriction was established by either the application of endothelin-1 or the thromboxane A2 mimetic U46619. This was followed by an intravascular or aerosol application of BNP. CGMP was measured in the recirculating buffer fluid using a radioimmunoassay technique. Results During a stable plateau of U46619 induced pulmonary vasoconstriction (mean pulmonary artery pressure, PAP 25.5 ± 0.23 mmHg), the intravascular administration of BNP induced a rapid vasodilation (mean PAP 18.13 ± 0.95 mmHg, p

Pulmonary hypertension (PH) leads to an increased right ventricular workload, cardiac failure and death. In idiopathic pulmonary arterial hypertension (PAH) the vasodilating vasoactive intestinal peptide (aviptadil) is deficient. The aim of the present study was to test the acute effects on haemodynamics and blood gases, and the safety, of a single dose of inhaled aviptadil in chronic PH. A total of 20 patients with PH (PAH in nine, PH in lung disease in eight and chronic thromboembolic PH in three) inhaled a single 100-microg dose of aviptadil during right-heart catheterisation. Haemodynamics and blood gases were measured. Aviptadil aerosol caused a small and temporary but significant selective pulmonary vasodilation, an improved stroke volume and mixed venous oxygen saturation. Overall, six patients experienced a pulmonary vascular resistance reduction of >20%. In patients with significant lung disease, aviptadil tended to improve oxygenation. The pulmonary vasodilating effect of aviptadil aerosol was modest and short-lived, did not cause any side-effects and led to a reduced workload of the right ventricle without affecting systemic blood pressure. Aviptadil inhalation tended to improve oxygenation in patients with significant lung disease. Further studies are needed to evaluate the full therapeutic potential of aviptadil aerosol, including higher doses and chronic treatment.

Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

The 1st step in the posttranslational hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine] modification of eukaryotic translation initiation factor 5A (eIF5A) is catalyzed by deoxyhypusine synthase (DHS). The eIF5A intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DHH), thereby converting the eIF5A precursor into a biologically active protein. Depletion of eIF5A causes inhibition of cell growth, and the identification of eIF5A as a cofactor of the HIV Rev protein turns this host protein and therefore DHS into an interesting target for drugs against abnormal cell growth and/or HIV replication. The authors developed a 96-well format DHS assay applicable for the screening of DHS inhibitors. Using this assay, they demonstrate DHS inhibition by AXD455 (Semapimod, CNI-1493). This assay represents a powerful tool for the identification of new DHS inhibitors with potency against cancer and HIV.

Background: Mast cells (MCs) are multifunctional effector cells of the immune system. These cells originate from pluripotent hemopoietic progenitors. In contrast to basophils and other leukocytes, MCs exhibit a remarkably long life span (years) in vivo. Although a role for stem cell factor (SCF) and SCF receptor (KIT) in long-term survival of MCs has been proposed, the underlying biochemical mechanisms remain unknown. Materials and Methods:We have examined expression of ‘survival-related’ molecules of the bcl-2 family including bcl-2 and bcl-xL, in primary human MCs and the human MC line HMC-1. Primary MCs were isolated from dispersed lung tissue by cell sorting using an antibody against KIT. mRNA expression was analyzed by RT-PCR and Northern blotting. Results: As assessed by RT-PCR, purified unstimulated lung MCs (>98% pure) exhibited KIT- and bcl-xL mRNA, but did not express bcl-2 mRNA. However, exposure of lung MCS to SCF (100 ng/ml) for 8 h resulted in expression of bcl-2 mRNA. Corresponding results were obtained by immunocytochemistry. In fact, exposure of MC to SCF resulted in expression of the bcl-2 protein whereas unstimulated MCs displayed only the bcl-xL protein without expressing the bcl-2 protein. The human MC leukemia cell line HMC-1, which contains a mutated and intrinsically activated SCF receptor, showed constitutive expression of both bcl-2 and bcl-xL at the mRNA and protein level. Conclusion: Our data show that human MCs can express members of the bcl-2 family. It is hypothesized that bcl-xL plays a role in KIT-independent growth of MCs, whereas bcl-2 may be involved in KIT-dependent functions of MCs.

Recent data suggest that mast cells (MCs) in patients with systemic mastocytosis or mast cell leukemia express a CD2-reactive antigen. To explore the biochemical nature and function of this antigen, primary MCs as well as the MC line HMC-1 derived from a patient with mast cell leukemia were examined. Northern blot experiments revealed expression of CD2 messenger RNA in HMC-1, whereas primary nonneoplastic MCs did not express transcripts for CD2. In cell surface staining experiments, bone marrow (BM) MCs in systemic mastocytosis (n = 12) as well as HMC-1 cells (30%-80%) were found to express the T11-1 and T11-2 (but not T11-3) epitopes of CD2. By contrast, BM MCs in myelodysplastic syndromes and nonhematologic disorders (bronchiogenic carcinoma, foreskin phimosis, uterine myeomata ) were consistently CD2−. All MC species analyzed including HMC-1 were found to express LFA-3 (CD58), the natural ligand of CD2. To study the functional role of CD2 on neoplastic MCs, CD2+ and CD2− HMC-1 cells were separated by cell sorting. CD2+ HMC-1 cells were found to form spontaneous aggregates and rosettes with sheep erythrocytes in excess over CD2−cells, and a T11-1 antibody inhibited both the aggregation and rosette formation. Moreover, exposure of CD2+ HMC-1 cells to T11-1 or T11-2 antibody was followed by expression of T11-3. In addition, stimulation of neoplastic MCs through T11-3 and a second CD2 epitope resulted in histamine release. These data show that neoplastic MCs express functionally active CD2. It is hypothesized that expression of CD2 is associated with pathologic accumulation and function of MCs in systemic mastocytosis.

Dendritic cells (DCs), nature's adjuvant, must mature to sensitize T cells. However, although the maturation process is essential, it is not yet fully understood at the molecular level. In this study, we investigated the course of expression of the unique hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A), which is part of a particular RNA nuclear export pathway, during in vitro generation of human DCs. We show that eIF-5A expression is significantly upregulated during DC maturation. Furthermore, an inhibitor of the hypusine modification, GC7 (N1-guanyl-1,7-diaminoheptane), prevents CD83 surface expression by apparently interfering with nucleocytoplasmic translocation of the CD83 mRNA and, importantly, significantly inhibits DC-mediated T lymphocyte activation. The data presented suggest that CD83 mRNA is transported from the nucleus to the cytoplasm via a specific nuclear export pathway and that hypusine formation appears to be essential for the maturation of functional DCs. Therefore, pharmacological interference with hypusine formation may provide a new possibility to modulate DC function.

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine, a modification that appears to be required for cell proliferation. This hypusine-modified protein stimulates synthesis of methionyl-puromycin in an in vitro assay which mimics the formation of the first peptide bond during protein synthesis, although the exact role of eIF-5A in vivo is still unknown. The unexpected finding that eIF-5A is a cellular cofactor of the HIV-1 Rev trans-activator protein may, however, provide a novel opportunity to reveal precisely what function eIF-5A performs in eukaryotic cells. In this review article, we first present a brief description of HIV-1 Rev function, followed by an overview of the data that identified eIF-5A as a Rev cofactor and, finally, discuss novel findings with respect to cellular eIF-5A activities.

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 trans-activator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent—human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to threedifferent chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12–p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.

Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-alpha is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed.

A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additonal eIF-5A -related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A . The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication was demonstrated by using tat- and rev-directed antisense oligoribonucleotides 68 and 69 nucleotides in length. In this study, human T-lymphoid cells were transduced with a murine amphotropic retroviral vector containing a polymerase III-driven chimeric gene consisting of the human tRNA(imet) sequence and the short tat- and rev-directed antisense sequences that had been shown before to inhibit HIV-1 replication. Pools of transduced, G418-resistant human T-lymphoid Jurkat or CEM cells showed reduced replication of HIV-1 in the presence of antisense-containing chimeric transcripts, but not with sense sequence-containing transcripts. These results demonstrate that short inhibitory antisense RNA transcripts can be stably expressed endogenously using polymerase III promoters, which can reduce replication of HIV-1. The approach described in this work combines the advantages of short and, usually, synthetic oligonucleotides with the stable intracellular expression of inhibitory genes for HIV-1 in target cells. Considering the small size of the described chimeric polymerase III genes, it appears feasible to combine multiple antiviral genes with the currently available retroviral vectors as gene delivery systems.

Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 4.

The reaction of cyclosporin A (CsA) with Lawesson's reagent under different conditions yields various thiocyclosporins, in which carbonyl O-atoms and/or the hydroxy O-atom of the MeBmt residue are replaced by an S-atom. The position of the S-atom is determined by NMR spectroscopy, and the conformations of the products are studied by NMR spectroscopy and X-ray crystallography. Some of the thiocyclosporins show interesting conformational properties. Whereas one conformation strongly dominates for CsA in CDCL3, two conformers A and B, in a ratio 58:42 are found for [1ψ2, CS–NH]CsA. Extensive NMR studies including new 2D and 3D heteronuclear techniques and restrained MD calculations using ROE effects demonstrate that the major conformer A is identical to CsA, while the minor conformer B contains an additional cis peptide bond between the Sar3 and MeLeu4 residues. [4ψ5, CSNH; 7ψ8, CS–NH]CsA exhibits a conformation very similar to crystalline CsA. However, the D-Ala8NH, MeLeu6Co γ-turn H-bond is not present in this dithio analogue. Also different is the MeBmt1side-chain conformation, the dithio conformation showing a strong MeBmt1OH, Sar3CO H-bond. Immunosuppressive activities of thiocyclosporins are measured in IL-2 and IL-8 reporter gene assays. Their activities are discussed in relation to their conformations.

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by viral and cellular factors interacting with cis-elements located in the retroviral long terminal repeat (LTR). In this report we analyzed HIV-1 LTR-specific regulatory sequences responsive to the HIV-1 Tat and HTLV-I Tax trans-activator proteins. Our results indicate that the Sp1 binding sites in the HIV-1 LTR are crucially involved in Tat-mediated gene expression in human Jurkat T-cells whereas they are dispensable for HTLV-I Tax-induced activation. In contrast, the NF-kB binding sites within the HIV-1 LTR are essential for Tax-mediated transcription but had only marginal effect on Tat-induced reporter gene expression.

Cell recognition molecules play a crucial role in the regulation of immune cells. We recently found that mast cells (MCs) express leukocyte recognition molecules, including ICAM-1 antigen, a natural ligand of LFA-1. We here report that interleukin 4 (IL-4), a pleiotropic cytokine and mast cell differentiation factor, selectively promotes expression of surface ICAM-1 antigen and ICAM-1 mRNA in human MCs. IL-4 also up-regulates ICAM-1 antigen in cells of monocyte/macrophage lineage but has no effect on ICAM-1 antigen expressed on basophils, fibroblasts, or lymphocytes. The increase in expression of mast cell/macrophage ICAM-1 antigen induced by IL-4 may contribute to the accumulation of leukocytes and facilitate cell-contact-dependent regulation of immune cells in inflamed tissues.

S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, can be specifically inhibited by the experimental drug SAM486A. The pharmaceutical interference with SAMDC activity results in the depletion of the intracellular pool of spermidine and spermine. In particular, low spermidine levels compromise hypusine modification and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), which is a cellular cofactor of the essential human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev. In the present study, we show that SAM486A efficiently suppresses HIV-1 replication, including the replication of viruses that are resistant to multiple reverse transcriptase and protease inhibitors. At drug concentrations that efficiently inhibit the formation of progeny viruses, no toxic effects of SAM486A on cellular metabolism are observed. It is demonstrated that the antiretroviral effect of SAM486A is based on the fact that Rev activity is severely compromised in drug-treated cells. Thus, inhibition of cellular SAMDC activity may provide a novel strategy to achieve suppression of otherwise drug-resistant viruses.

The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1. However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41. Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options. Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy. Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS). By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein. We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells. We show that CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed. Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Helicobacter pylori has a major aetiological role in human gastric carcinogenesis but the cellular and molecular pathways by which infection promotes transformation remain to be resolved. This study demonstrates that H. pylori exposure to MKN-1, ST42, and MKN-28 gastric epithelial tumour cells results in the activation of HB-EGF gene expression and EGFR tyrosine phosphorylation. These cell responses are induced by both cagPAI positive and cagPAI negative H. pylori strains and are dependent on cell surface expression of the HB-EGF precursor. The induction of HB-EGF gene transcription by H. pylori requires metalloprotease-, EGFR-, and Mek1-activities, indicating the involvement of the "triple membrane passing signal" (TMPS) for EGFR transactivation. Moreover, the release of the inflammatory cytokine IL-8 by cells exposed to H. pylori is significantly impaired by inhibitors of TMPS pathway elements. Our findings support a model in which H. pylori triggers constitutive EGFR signal activation, which enhances IL-8 production, and initiates neoplastic transformation of gastric epithelial cells.

We introduced a methodology to efficiently exploit natural-language expressed biomedical knowledge for repurposing existing drugs towards diseases for which they were not initially intended. Leveraging on developments in Computational Linguistics and Graph Theory, a methodology is defined to build a graph representation of knowledge, which is automatically analysed to discover hidden relations between any drug and any disease: these relations are specific paths among the biomedical entities of the graph, representing possible Modes of Action for any given pharmacological compound. These paths are ranked according to their relevance, exploiting a measure induced by a stochastic process defined on the graph. Here we show, providing real-world examples, how the method successfully retrieves known pathophysiological Mode of Actions and finds new ones by meaningfully selecting and aggregating contributions from known bio-molecular interactions. Applications of this methodology are presented, and prove the efficacy of the method for selecting drugs as treatment options for rare diseases., 8 pages, 7 figures

Ribosomal protein L5 is part of the 60 S ribosomal subunit and localizes in both the cytoplasm and the nucleus of eukaryotic cells, accumulating particularly in the nucleoli. L5 is known to bind specifically to 5 S rRNA and is involved in nucleocytoplasmic transport of this rRNA. Here, we report a detailed analysis of the domain organization of the human ribosomal protein L5. We show that a signal that mediates nuclear import and nucleolar localization maps to amino acids 21–37 within the 297-amino acid L5 protein. Furthermore, carboxyl-terminal residues at positions 255–297 serve as an additional nuclear/nucleolar targeting signal. Domains involved in 5 S rRNA binding are located at both the amino terminus and the carboxyl terminus of L5. Microinjection studies in somatic cells demonstrate that a nuclear export signal (NES) that maps to amino acids 101–111 resides in the central region of L5. This NES is characterized by a pronounced clustering of critical leucine residues, which creates a peptide motif not previously observed in other leucine-rich NESs. Finally, we present a refined model of the multidomain structure of human ribosomal protein L5.

Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans - activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.

The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viral trans -regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis -acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form a trans -activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans -activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans -dominant (dominant-negative) fashion.

It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.

Recent data suggest that mast cells (MC) are involved in the regulation of leukocyte accumulation in inflammatory reactions. In this study, expression of leukocyte-chemotactic peptides (chemokines) in purified human lung MC (n = 16) and a human mast cell line, HMC-1, was analyzed. Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) showed baseline expression of monocyte chemoattractant protein (MCP)-1 mRNA in unstimulated MC. Exposure of MC to recombinant stem cell factor (rhSCF, 100 ng/mL) or anti-IgE (10 μg/mL) was followed by a substantial increase in expression of MCP-1 mRNA. Neither unstimulated nor stem cell factor (SCF )-stimulated lung MC expressed transcripts for interleukin-8 (IL-8), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, or RANTES by Northern blotting. The mast cell line HMC-1, which contains a mutated and intrinsically activated SCF-receptor, was found to express high levels of MCP-1 mRNA in a constitutive manner. Exposure of HMC-1 cells to rhSCF resulted in upregulation of MCP-1 mRNA expression, and de novo expression of MIP-1β mRNA. The SCF-induced upregulation of MCP-1 mRNA in lung MC and HMC-1 was accompanied by an increase in immunologically detectable MCP-1 in cell supernatants (sup) (lung MC [

The urokinase receptor system is involved in several biological processes including extracellular proteolysis, cell invasion, and chemotaxis. Mast cells are multifunctional perivascular cells that play an important role in the regulation of microenvironmental events. We report that primary human mast cells and the human mast cell line HMC-1 express the receptor for urokinase. As assessed by Northern blotting and reverse transcription polymerase chain reaction technique, purified human lung mast cells and HMC-1 cells expressed urokinase receptor mRNA in a constitutive manner. Using a toluidine blue/immunofluorescence double staining technique and monoclonal antibodies, surface expression of urokinase receptor was demonstrable in lung, skin, uterus, heart, and tonsil mast cells, whereas the low density lipoprotein receptor-related protein was not detectable. Binding of monoclonal antibody VIM5 (recognizing the urokinase binding domain of urokinase receptor) to HMC-1 could be blocked by high molecular weight but not low molecular weight urokinase. Binding analyses performed with 123I-urokinase revealed expression of 271,000 +/- 55,000 high affinity urokinase binding sites per HMC-1 cell, with a calculated dissociation constant of 1. 29 +/- 0.3 nM. Purified urokinase induced dose-dependent migration of primary mast cells and HMC-1 in a chemotaxis assay without inducing release of histamine. The mast cell agonist stem cell factor also induced migration of HMC-1 and caused up-regulation of expression of urokinase receptor mRNA. Together, our data show that human mast cells express functional receptors for urokinase. Expression of urokinase receptors on mast cells may have implications for mast cell-dependent microvascular processes associated with fibrinolysis, migration, or local tissue repair.

Previously, we described two mutants of the cellular Rev co-factor, eukaryotic initiation factor 5A (eIF-5A M13 and M14), which suppress human immunodeficiency virus type 1 (HIV-1) SF2 replication in clonal T cell lines. This study introduced the notion that it is possible to develop gene therapies against infectious agents on the basis of mutant host factors required for viral replication. In this report, we provide further evidence to support this new paradigm and describe murine leukemia virus (MLV)-based retroviral vectors expressing three different eIF-5A mutants from the viral long terminal repeat (LTR). HIV-1 replication (SF2, HXB-3) was reduced up to 2 orders of magnitude in transduced, polyclonal T cell populations. All eIF-5A mutants also showed antiviral activity (approximately seven-fold reduction) in a chronic HIV-1 infection model. Expression of eIF-5A mutant M13 delta in peripheral blood lymphocytes (PBLs) showed no difference in proliferation and metabolic activity as determined in a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)-assay, suggesting that expression of this type of mutant protein is not associated with cellular toxicity. In summary, these data suggest that gene therapy for HIV-1 infection can be developed on the basis of mutants of the Rev co-factor eIF-5A.

Eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor required for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element: Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF-5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent-human cell hybrids we localized the deoxyhupusine synthase gene to human chromosome 19.

The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.

We have previously reported that chimeric neomycin phosphotransferase (neo)-Rev response element (RRE) transcripts suppress the function of the human immunodeficiency virus type 1 (HIV-1) Rev trans-activator protein in HeLa cells. In an extension of these experiments, human CD4+ CEM cells (G418-resistant cell populations and clonal isolates) stably expressing chimeric neo-RRE genes (2, 3, or 6 RRE copies) were generated using retroviral-mediated gene transfer. The transduced CEM clones were infected with the HIV-1 HTLVIIIB isolate and the following three phenotypes were observed: (i) the transduced CEM cells were readily infected with HIV-1 indistinguishable from the control CEM cells; (ii) the appearance of HIV-1 replication markers was significantly delayed; (iii) no signs of HIV-1 replication were detectable although proviral HIV-1 DNA sequences could be detected in these cells. Furthermore, HIV antigen expression was limited in neo-resistant CEM cell populations inoculated with the HIV-1 HTLVIIIB isolate. Only 10% of the CEM-pX17-3xRRE cells and 20% of the CEM-pX17-2xRRE cells displayed HIV-1 antigens 43 days after challenge and had retained CD4 surface expression on 47% and 64% of the cells, respectively. In sharp contrast, 80% of the CEM-pX17 or the CEM-pX17-6xRRE cells expressed HIV-1 antigens but no CD4 antigens were detectable in these cultures. These results clearly indicate that RRE decoys could be developed into an effective somatic gene therapy approach against HIV-1 induced acquired immunodeficiency syndrome (AIDS).

Human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Currently, no satisfactory treatment for this viral disease is available. Somatic gene therapy has been proposed as an alternative to conventional therapies. Several antiviral gene therapy approaches including ribozymes, antisense inhibition, and RNA-decoy strategies, as well as dominant-negative mutants of HIV-1 proteins (Gag, Tat, and Rev) have been suggested. To prove the concept of trans-dominant inhibition of HIV-1 replication, we transduced CEM cells with a retroviral vector encoding a dominant-negative rev gene. Amplification of integrase-specific proviral sequences from high molecular weight DNA indicated successful HIV-1 human T-lymphotropic virus type IIIB (HTLV-IIIB) infection of all cells. In contrast to CEM cells and CEM cells expressing the rev wild-type (wt) gene, infection of two CEM-RevM10 clones with HIV-1 did not result in the release of significant levels of p24 Gag antigen as measured by antigen capture assay, indicating a block in HIV-1 replication due to the presence of the trans-dominant Rev protein. Furthermore, the parental CEM cells as well as CEM cells expressing the Rev wt protein were effectively killed in the course of the HIV-1 infection, whereas all CEM cells expressing the RevM10 protein were unaffected in their growth rate.

Expression of the Q5k gene was examined by northern blot analysis and polymerase chain reaction (PCR) in the AKR mouse and various cell lines, each of the H-2k haplotype. Our results show that Q5k mRNA is present during the whole postimplantational development of the AKR embryo/fetus (gestation day 6 to 15). In the juvenile mouse (week 2 to 4) transcription of the Q5k gene persisted in all organs examined. In contrast, in the adult animal expression of the Q5k gene was limited to the thymus and uterus of the pregnant mouse. Upon malignant transformation, the amount of Q5k-specific mRNA increased dramatically in thymus and could also be observed in the spleen of thymoma bearing animals. Expression of the Q5k gene was also detectable in several transformed mouse cell lines. Mitogen stimulation or treatment with cytokines induced Q5k expression in primary spleen cell cultures. A possible explanation for the tissue-restricted expression in the adult AKR mouse is discussed.

Using an oligodeoxynucleotide generated by rapid PCR amplification of 5′-cDNA ends (5′-RACE) as a detection probe, we have isolated a new genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A). Sequence analysis revealed that the eIF-5A coding region is identical to the corresponding cDNA but interrupted by three introns. In a plasmid shuffle experiment we show functional replacement of the essential homologous gene in Saccharomyces cerevisiae by this human eIF-5A .

In a series of newly isolated AKR leukemias, some tumors expressed large amounts of both H-2K and H-2D molecules, while others had reduced levels of both antigens. The number of H-2 antigens expressed in steady state showed a consistent correlation with the rate of synthesis of the H-2 class I heavy chain and the beta-2 microglobulin (beta 2m) light chain, and with the amount of H-2- and beta 2m-specific mRNA present in the tumors. Stimulation of leukemic cells with interferon induced an increased transcription of both H-2 and beta 2m mRNA. These results suggest that there is a mechanism that regulates, at the transcriptional level, the coordinate expression of H-2K and H-2D heavy chains, and the beta 2m light chains encoded by genes on chromosomes 17 and 2, respectively.

The Q4 class I gene has been shown to participate in gene conversion events within the mouse major histocompatibility complex. Its complete genomic nucleotide sequence has been determined. The 5' half of Q4 resembles H-2 genes more strongly than other Q genes. Its 3' end, in contrast, is Q-like and contains a translational stop signal in exon 5 which predicts a polypeptide with an incomplete membrane spanning segment. The presence of two inverted B1 repeats suggests that part of the Q4 gene may be mobile within the genome. Gene transfer experiments have shown that the Q4 gene encodes a beta 2-microglobulin associated polypeptide of Mr 41,000. A similar protein was found in activated mouse spleen cells. The Q4 polypeptide was found to be secreted both by spleen cells and by transfected fibroblasts and was not detectable on the cell surface. Antibody binding and two-dimensional gel electrophoresis indicate that the Q4 molecule is identical to a mouse class I polypeptide, Qb-1, which has been previously described.

Complementary DNA (cDNA) probes were used to investigate the extrahepatic production of the major histocompatibility complex (MHC)-linked complement components C4, factor B and C2 in various normal human tissues. The presence of the corresponding messenger RNA (mRNA) was tested by Northern blot analysis. Complement C4 mRNA was found in liver, and with high intensity also in normal kidneys. In contrast, no C2 mRNA and only very low amounts of factor B mRNA could be detected in the kidney. Slot blot hybridization was performed to quantitate the amount of C4 mRNA, and the intensity of C4 mRNA hybridization in the kidney samples was about 25% compared with liver RNA. C4-specific transcripts were not present in isolated glomeruli but in the renal interstitium. Other human tissues, such as tonsil, spleen, thymus, brain, lung and peripheral mononuclear cells, contained no C4 mRNA. Low amounts of C4 mRNA were found in colon, thyroid gland, lymph node and breast carcinoma. The results obtained with lung, where C2 mRNA was found but no C4 mRNA, further indicate an independent, tissue-specific regulation of the class III gene expression. The results, showing that the complement C4 genes are transcribed very efficiently in normal human kidney, suggest a direct role of complement C4 in renal pathogenesis.

SUMMARY We established the organization of the AKR Qa region and determined the sequence of the 44 and Q5 genes. Restriction mapping and genomic Southern blot analysis revealed that the AKR strain codes for only three H-2K homologous genes in this region. The AKR Q5 gene is not homologous to the Q5 gene of the C57BL strain, but is presumably allelic to the Q5 gene isolated from Balb/c. The organization and structure of the AKR Qa family is virtually identical to the Qa genes of the C3H mouse. The AKR Q5 gene, in contrast to other H-2K homologous Qa region genes, codes for a typical transmembrane region, and upon transfection into BHK cells, a 1.6 kb Q5 transcript is detected.

Inflammatory mediators such as tumor necrosis factor (TNF) or interleukin-1 (IL-1) and bacterial lipopolysaccharides (LPS) were shown to shift the hemostatic balance of the endothelial cell (EC) surface in favor of procoagulant activities by inducing tissue factor (TF) expression and downregulation of thrombomodulin (TM). In the present study, the effects of IL-4 on these regulatory mechanisms were investigated using cultured human umbilical vein EC. TM downregulation induced by the pyrogens IL-1 (100 U/mL), TNF (500 U/mL), and LPS (20 micrograms/mL) to less than 50% of TM activity of untreated cells during a 12-hour incubation period was completely neutralized when these mediators were coincubated with IL-4 (100 U/mL). In accordance with TM surface activity, TM messenger RNA was decreased by IL-1, TNF, and LPS to less than 40% of untreated cells; this effect was in part antagonized by IL-4. No influence of IL-4 on EC tissue factor induction by IL-1, TNF, and LPS was found. Binding studies using 125I- radiolabeled IL-4 suggest that EC express a single class of high- affinity binding sites (kd = 3.2 pmol/L; 2,000 to 2,500 receptors per cell). These results show that IL-4, in part, protects the EC surface against pyrogen-induced procoagulant changes. Transcriptional regulatory mechanisms seem to be involved in EC surface TM regulation.