BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride], a nonimidazole inverse agonist/antagonist at the human histamine H3 receptor: Preclinical pharmacology.

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity approximately 50% higher than that of ciproxifan. Its in vitro potency was approximately 6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways.

DENN domains are found in a variety of signaling proteins but their exact function remains undefined. Some of the DENN-containing proteins, such as rat Rab3GEP (Rab3 GDP/GTP exchange protein) or mouse Rab6IP1 (Rab6 interacting protein 1) interact with GTPases of the Rab family. Others, such as human MADD (MAP (Mitogen-activated protein) kinase activating protein containing death domain) and human ST5 (Suppressor of tumoreginicity 5) gene products are involved in regulation of MAPKs (Mitogen-activated protein kinases) signaling pathways. Using a combination of profile-based and bidimensional analyses, we show here that DENN domains are much larger than described to date in domain databases, always encircled on both sides by more divergent domains, that we called uDENN and dDENN. These however share conserved amino acids which could play a key role in the DENN functions.

hKv4.3 channel characterization and regulation by calcium channel antagonists.

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.

Functional specificity conferred by the unique plasticity of fully alpha-helical Ras and Rho GAPs.

Structural comparisons of the two GTPase activating proteins (GAPs) p120 and p50 in complex with Ras and Rho, respectively, allowed us to decipher the functional role of specific structural features, such as helix alpha8c of p120 and helix A1 of p50, necessary for small GTPase recognition. We identified important residues that may be critical for stabilization of the GAP/GTPase binary complexes. Detection of topohydrophobic positions (positions which are most often occupied by hydrophobic amino acids within a family of protein domains) conserved between the two GAP families led to the characterization of a common flexible four-helix bundle. Altogether, these data are consistent with a rearrangement of several helices around a common core, which strongly supports the assumption that p50 and p120 GAPs derive from a unique fold. Considered as a whole, the remarkable plasticity of GAPs appears to be a means used by nature to accurately confer functional specificity.

Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current.

OBJECTIVE: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. METHODS: The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. RESULTS: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to -40 mV and the steady-state inactivation curve had a midpoint of -47.4 +/- 0.3 mV and a slope of 5.9 +/- 0.2 mV. Rat ventricular Ito current was activated at potentials positive to -20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of -29.1 +/- 0.7 mV and 4.5 +/- 0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2 +/- 4.1 msec and 23. +/- 1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54 +/- 0.13 mM and 0.14 +/- 0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogeneous. CONCLUSION: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogeneous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current.

Sequence and 3D structural relationships between mammalian Ras- and Rho-specific GTPase-activating proteins (GAPs): the cradle fold.

An extensive study of both sequence and recent 3D structural data concerning GTPase interacting domains of Ras- and Rho-specific GTPase-activating proteins (GAPs) shows that these two subfamilies share a same 3D scaffold and are thus related to each other. This relationship has heretofore remained undetected although these domains of similar size are both totally alpha-helical and activate nearly structurally identical targets (Ras and Rho proteins). In this report, sequence similarities correlated to 3D structures of p120rasGAP and p50rhoGAP were detected using the sensitive two-dimensional method hydrophobic cluster analysis (HCA). These patterns were further extended to other members in each subfamily and the geometry orientation of crucial arginines R789 in p120 and R282 in p50 and of important stabilizing residues like p120R903 and p50N391 was confirmed. This overall structural relationship is centered on an invariant motif of three consecutive helices that we suggest to name the 'cradle fold'. This observation opens new perspectives to understand how small GTPases are specifically regulated.

[Experimental models of angiogenesis for the study of the Ets1 transcription factor]. / Modèles expérimentaux d'angiogenèse pour l'étude du facteur de transcription Ets1.

The Ets1 transcription factor gene is expressed in endothelial cells during blood vessel formation under normal or pathological conditions. The proposed hypothesis aims to involve Ets1 in the regulation of angiogenesis processes by activating the transcription of genes encoding matrix-degrading proteases. In vivo experiments allowing specific manipulation of ets1 gene expression or activity within endothelial cells are necessary to investigate the functional role of Ets1. Two experimental models using retroviruses expressing ets1 mutants have been chosen: Chicken embryo development and tumour-induced angiogenesis in mice. Another approach targeted on the vascular endothelium has been developed in order to obtain transgenic mice expressing specifically in endothelial cells an ets1 transdominant negative mutant under the control of the tek and tie promoters. These tools should allow interference with Ets1 activity at defined stages during normal or pathological development.

Genesis of discrete higher order DNA fragments in apoptotic human prostatic carcinoma cells.

Higher order DNA fragmentation may be an essential signal in apoptosis. We found that etoposide (VP-16) induced apoptosis in human DU-145 prostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells detached from the monolayer; untreated or VP-16-treated attached cells retained a normal morphology. We describe a radiolabeled alu-I sequence-based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecular weight DNA fragments in detached (apoptotic) and attached (preapoptotic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were > or = 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that these DNA fragments increased 9 +/- 2-, 8 +/- 2-, and 25 +/- 11-fold versus control, respectively, with a 24-hr exposure to 30 microM VP-16 in attached cell populations. In detached VP-16-treated cells, there was accrual of 30-50-kbp DNA fragments with a concomitant loss of the > or = 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weight DNA fragmentation was inhibited by cycloheximide treatment and was common to other apoptotic agents, including melphalan and bleomycin. These findings suggest that the > or = 1-mbp and 450-600-kbp DNA fragments are products of endonuclease activation and are not topoisomerase II/DNA interactions. Finally, the generation of the 30-50-kbp DNA fragments may mediate chromatin condensation, which characterizes apoptosis.

Yeast cysteine proteinase gene ycp1 induces resistance to bleomycin in mammalian cells.

Tumor resistance to the glycopeptide anticancer drug bleomycin (BLM) has been suggested to involve metabolic inactivation by BLM hydrolase. Direct evidence for this hypothesis is lacking due to difficulties in obtaining full-length BLM hydrolase cDNA from mammalian cells. In the present investigation, we used the yeast cysteine proteinase gene ycp1, a homologue of the mammalian BLM hydrolase gene, to provide direct evidence of the importance of BLM metabolism in BLM resistance. Transfection of ycp1 into NIH 3T3 cells induced resistance of these cells to BLM. The ycp1-transfected cells also metabolized BLM A2 to its inactive metabolite deamido-BLM A2 to a much greater extent. The ycp1-induced BLM resistance was completely reversed by the cysteine proteinase inhibitor E-64, a known inhibitor of BLM hydrolase. Transfection of NIH 3T3 cells with the plasmid pUT533-Sh ble, a bacterial BLM resistance gene that encodes a 14-kDa protein that does not metabolize BLM, also induced BLM resistance, but this resistance was not overcome by E-64. The results demonstrate that increased BLM hydrolase activity in NIH 3T3 cells causes BLM resistance and that inhibition of BLM metabolism sensitizes these cells to BLM. Thus, the molecular approach described in the present study directly implicates BLM hydrolase in BLM resistance.

Invasive tumors induce c-ets1 transcription factor expression in adjacent stroma.

The stroma reaction plays a central role in tumor growth, invasion and metastasis. Tumor growth is dependent on angiogenesis and requires the vascular supply provided by new capillary blood vessels of the stroma. The expression of the gene encoding the transcription factor c-ets1 is localized within fibroblasts and endothelial cells of the stromal compartment. This expression correlates with the accumulation of transcripts for potential target genes such as collagenase I and stromelysin I in stromal fibroblasts surrounding malignant cells in invasive tumors. We suggest that c-Ets1 protein might regulate the transcription of the genes coding for matrix-degrading proteases necessary for both angiogenesis and tumor invasion.

Gene therapy and endothelial cell targeting for cancer.

The endothelium represents a potentially critical target for gene therapy because of its anatomical location and its importance in the viability in both normal and malignant tissues. Protecting the endothelium of normal tissues, such as the lungs, from the toxic effects of current antineoplastic agents and the destruction of the tumor vasculature are reasonable goals. As a target, however, the endothelium continues to represent a significant challenge. While gene delivery to cultured endothelial cells is possible, improved delivery systems are required, as well as cell-specific promoters, before in vivo gene therapy to important endothelial populations can be accomplished.

Nuclear localization of bacterial Streptoalloteichus hindustanus bleomycin resistance protein in mammalian cells.

Prokaryotes produce a variety of toxins that affect genomic function of both eukaryotes and prokaryotes. The 375-base pair bacterial gene Streptoalloteichus hindustanus (Sh) ble encodes a small protein, Streptoalloteichus hindustanus bleomycin resistance protein (BRP), that inhibits in vitro DNA cleavage by the prokaryotic glycopeptide bleomycin, which is a clinically used anticancer drug. NIH/3T3 cells infected with a retroviral vector containing Sh ble (SH-9 cells) were highly resistant to the cytotoxicity of bleomycin-like drugs but not to the cytotoxicity of other, structurally unrelated, DNA-cleaving agents. Expression of BRP did not markedly alter total cellular content or distribution of bleomycin-like compounds. Fluorescently labeled bleomycin was primarily localized in cytoplasmic vesicles in NIH/3T3 and SH-9 cells, whereas BRP, which has no established nuclear localization sequence, was segregated to the nucleus and more specifically to euchromatin. This karyophilic BRP may intercept bleomycin in the nucleus.

Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.