3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors

Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutagenesis experiments.     

Seek & Destroy, use of targeting peptides for cancer detection and drug delivery

Accounting for 16 million new cases and 9 million deaths annually, cancer leaves a great number of patients helpless. It is a complex disease and still a major challenge for the scientific and medical communities. The efficacy of conventional chemotherapies is often poor and patients suffer from off-target effects. Each neoplasm exhibits molecular signatures – sometimes in a patient specific manner – that may completely differ from the organ of origin, may be expressed in markedly higher amounts and/or in different location compared to the normal tissue. Although adding layers of complexity in the understanding of cancer biology, this cancer-specific signature provides an opportunity to develop targeting agents for early detection, diagnosis, and therapeutics. Chimeric antibodies, recombinant proteins or synthetic polypeptides have emerged as excellent candidates for specific homing to peripheral and central nervous system cancers. Specifically, peptide ligands benefit from their small size, easy and affordable production, high specificity, and remarkable flexibility regarding their sequence and conjugation possibilities. Coupled to imaging agents, chemotherapies and/or nanocarriers they have shown to increase the on-site delivery, thus allowing better tumor mass contouring in imaging and increased efficacy of the chemotherapies associated with reduced adverse effects. Therefore, some of the peptides alone or in combination have been tested in clinical trials to treat patients. Peptides have been well-tolerated and shown absence of toxicity. This review aims to offer a view on tumor targeting peptides that are either derived from natural peptide ligands or identified using phage display screening. We also include examples of peptides targeting the high-grade malignant tumors of the central nervous system as an example of the complex therapeutic management due to the tumor’s location. Peptide vaccines are outside of the scope of this review.     

Identification of novel p53 pathway activating small-molecule compounds reveals unexpected similarities with known therapeutic agents

Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents.     

Two distinct regions in the model protein Peb1 are critical for its heterologous transport out of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli is frequently the first-choice host organism in expression of heterologous recombinant proteins in basic research as well as in production of commercial, therapeutic polypeptides. Especially the secretion of proteins into the culture medium of E. coli is advantageous compared to intracellular production due to the ease in recovery of the recombinant protein. Since E. coli naturally is a poor secretor of proteins, a few strategies for optimization of extracellular secretion have been described. We have previously reported efficient secretion of the diagnostically interesting model protein Peb1 of Campylobacter jejuni into the growth medium of Escherichia coli strain MKS12 (ΔfliCfliD). To generate a more detailed understanding of the molecular mechanisms behind this interesting heterologous secretion system with biotechnological implications, we here analyzed further the transport of Peb1 in the E. coli host.     

A new model for the evolution of carnivory in the bladderwort plant (utricularia): adaptive changes in cytochrome C oxidase (COX) provide respiratory power

The evolution of carnivorous plants has been modeled as a selective tradeoff between photosynthetic costs and benefits in nutrient-poor habitats. Although possibly applicable for pitfall and flypaper trappers, more variables may be required for active trapping systems. Bladderwort (utricularia) suction traps react to prey stimuli with an extremely rapid release of elastic instability. Trap setting requires considerable energy to engage an active ion transport process whereby water is pumped out through the thin bladder walls to create negative internal pressure. Accordingly, empirical estimates have shown that respiratory rates in bladders are far greater than in leafy structures. Cytochrome C oxidase (COX) is a multi-subunit enzyme that catalyzes the respiratory reduction of oxygen to water and couples this reaction to translocation of protons, generating a transmembrane electrochemical gradient that is used for the synthesis of adenosine triphosphate (ATP). We have previously demonstrated that two contiguous cysteine residues in helix 3 of COX subunit I (COX I) have evolved under positive Darwinian selection. This motif, absent in approximately 99.9 % of databased COX I proteins from eukaryotes, Archaea, and Bacteria, lies directly at the docking point of COX I helix 3 and cytochrome C. Modeling of bovine COX I suggests the possibility that a vicinal disulfide bridge at this position could cause premature helix termination. The helix 3-4 loop makes crucial contacts with the active site of COX, and we postulate that the C-C motif might cause a conformational change that decouples (or partly decouples) electron transport from proton pumping. Such decoupling would permit bladderworts to optimize power output (which equals energy times rate) during times of need, albeit with a 20 % reduction in overall energy efficiency of the respiratory chain. A new model for the evolution of bladderwort carnivory is proposed that includes respiration as an additional tradeoff parameter.     

Serological survey for viral pathogens in Turkish rodents

Wild rodents (n = 330) were trapped around the villages of Altindere and Co?andere (Ma?ka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihem?in, Rize Province), and Bozdag (Odemi?, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.     

Two new eimerians (Apicomplexa) from insectivorous mammals in Madagascar

Fecal samples from 126 insectivorous mammals in Madagascar were collected between spring 1999 and fall 2001. In the Afrosoricida, 21 species in 5 genera were sampled, including 17 species of Microgale (31/96, 32% infected), Hemicentetes semispinosus (1/2, 50%), Oryzorictes hova (1/5, 20%), Setifer setosus (8/13, 61.5%), and Tenrec ecaudatus (5/8, 62.5%); in the Soricomorpha, only Suncus murinus was examined and 1/2 (50%) were infected. Two morphotypes of eimeriid oocysts, representing 2 presumptive new species, were found in 47 (37%) infected animals; only 2 afrosoricid hosts (2% of all hosts, 4% of infected hosts) had both oocyst morphotypes. Sporulated oocysts of the first morphotype, Eimeria tenrececaudata n. sp., are subspheroidal, 18.8 × 17.4 (17-22 × 15-20), with a length∶width ratio (L/W) of 1.1 (1.0-1.2); they lack a micropyle but may contain 0-2 polar granules and a single, small round oocyst residuum, 3 × 2.3. Sporocysts are lemon-shaped, 9.9 × 6.6 (9-11 × 5-8), with a L/W of 1.5 (1.2-2.0); they have a prominent, slightly flattened Stieda body and a substieda body but lack a parastieda body. The sporocyst residuum consists of only a few granules between the sporozoites, which are sausage-shaped and have a large posterior refractile body. Oocysts of the second morphotype, Eimeria setifersetosa n. sp. are spheroidal to subspheroidal, 30.1 × 28.6 (27-34 × 25-34), with a L/W of 1.1 (1.0-1.2); they lack both micropyle and oocyst residuum, but 1-2 polar granules are usually present. Sporocysts are subspheroidal to broadly ellipsoidal, 9.6 × 7.3 (9-11 × 6-8), with a L/W of 1.3 (1.1-1.7); they have a broad Stieda body, lack sub- and parastieda bodies, and have a residuum of a few granules scattered throughout the sporocyst. Sporozoites were not clearly defined, but what seemed to be a single large refractile body is seen, presumably in each sporozoite.     

Coxsackievirus B3-induced cellular protrusions: structural characteristics and functional competence

Virus-induced alterations in cell morphology play important roles in the viral life cycle. To examine the intracellular events of coxsackievirus B3 (CVB3) infection, green monkey kidney (GMK) cells were either inoculated with the virus or transfected with the viral RNA. Various microscopic and flow cytometric approaches demonstrated the emergence of CVB3 capsid proteins at 8 h posttransfection, followed by morphological transformation of the cells. The morphological changes included formation of membranous protrusions containing viral capsids, together with microtubules and actin. Translocation of viral capsids into these protrusions was sensitive to cytochalasin D, suggesting the importance of actin in the process. Three-dimensional (3D) live-cell imaging demonstrated frequent contacts between cellular protrusions and adjacent cells. Markedly, in spite of an increase in the cellular viral protein content starting 8 h postinfection, no significant decrease in cell viability or increase in the amount of early apoptotic markers was observed by flow cytometry by 28 h postinfection. Comicroinjection of viral RNA and fluorescent dextran in the presence of neutralizing virus antibody suggested that these protrusions mediated the spread of infection from one cell to another prior to virus-induced cell lysis. Altogether, the CVB3-induced cellular protrusions could function as a hitherto-unknown nonlytic mechanism of cell-to-cell transmission exploited by enteroviruses.     

6-O- and N-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.