Ligand Binding Characteristics of the Human Serotonin<Subscript>1<Emphasis Type="Italic">A</Emphasis></Subscript> Receptor Heterologously Expressed in CHO Cells

The serotonin_1A (5-HT_1A) receptors are important members of the superfamily of seven transmembrane domain G-protein coupled receptors. They appear to be involved in various behavioral, cognitive and developmental functions. Mammalian cells in culture heterologously expressing membrane receptors represent convenient systems to address problems in receptor biology. We report here the pharmacological characterization of one of the first isolated clones of CHO cells stably expressing the human 5-HT_1A receptor using the selective agonist 8-OH-DPAT and antagonist p-MPPF. In addition, we demonstrate that agonist and antagonist binding to the receptor exhibit differential sensitivity to the non-hydrolyzable GTP analogue, GTP--S, as was observed earlier with the native receptor from bovine hippocampus. These results show that the human 5-HT_1A receptor expressed in CHO cells displays characteristic features found in the native receptor isolated from bovine hippocampus and promises to be a potentially useful system for future studies of the receptor.These authors have contributed equally to the work     

Origin of a new Reticulitermes termite (Isoptera, Rhinotermitidae) inferred from mitochondrial and nuclear DNA data

The Holoarctic termite genus Reticulitermes is widely distributed in Europe. A new Reticulitermes species, R. sp. nov, was recently found in France and Italy. Its phylogenetic position was investigated using a 743-bp fragment of mitochondrial 16S rRNA-ND1 genes and 382-bp of the nuclear ITS2 region. Phylogenies for these sequences were estimated by neighbor-joining, maximum-parsimony and maximum-likelihood analysis. The results strongly supported a relationship between R. sp. nov. and the termite species from the eastern Mediterranean area including Reticulitermes balkanensis from the Balkans, Reticulitermes lucifugus from Turkey and Reticulitermes clypeatus from Israel. The hypothesis of a relationship between R. sp. nov. and the Japanese Reticulitermes speratus was rejected by parametric bootstrap. The current distribution of R. sp. nov. could be linked to postglacial colonization routes between Balkan refuge and northern regions.     

Effect of age and sex on the production of internal and external hydrocarbons and pheromones in the housefly, Musca domestica

The epicuticular and internal waxes of male and female houseflies were examined by capillary gas chromatography-mass spectrometry at closely timed intervals from emergence until day-6 of adulthood. New components identified included tricosan-10-one, 9,10-epoxyheptacosane, heptacosen-12-one, a series of odd-carbon numbered dienes from C31 to C39, several positional isomers of monoenes including (Z)-9- and 7-pentacosene and a number of methyl- and dimethylalkanes. (Z)-9-tricosene appears in internal lipids prior to appearing on the surface of the insect, suggesting that it is transported in the hemolymph to its site of deposition on the epicuticle. The large increases in the amount of (Z)-9-tricosene in females from day-2 until day-6 is compensated for by a concomitant decrease in (Z)-9-heptacosene. The C23 epoxide and ketone only appear in females after the production of (Z)-9-tricosene is induced, and are only abundant in epicuticular waxes, suggesting they are formed after (Z)-9-tricosene is transported to the cells which are involved in taking them to the surface of the insect. Mathematical analysis indicated that the time shift between internal production and external accumulation in females is more than 24 h. The divergence between male and female lipid production occurs at an early stage, when insects are less than one day old.     

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center,Rio de Janeiro,Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.     

Farnesyl transferase inhibitors: one target may be found in another

The fact that proteins such as Ras require farnesylation to induce malignant transformation prompted many investigators to design farnesyl transferase inhibitors (FTI) as novel anticancer drugs. FTIs inhibit the growth of ras transformed cells in vitro and induce tumor regression in ras dependent tumor in vivo. Moreover, FTIs inhibit tumor progression in human tumor xenograft models. Currently, FTIs are undergoing phase I and II trials in various cancer types. They show impressive antitumour efficacy and they lack toxicity. Despite these promising results, the development of such molecules in hindered by the absence of appropriate clinical endpoints and of surrogate biological markers. Indeed, it seems likely that Ras is not the critical target of FTIs and that inhibition of the farnesylation of proteins such as RhoB, might also contribute to the observed antitumour properties. Identification of targets that underlie their biological effect is essential in order to predict and evaluate their efficacy.     

Inward and outward potassium currents through the same chimeric human Kv channel

Voltage-gated ion channels are among the most intensely studied membrane proteins today and a variety of techniques has led to a basic mapping of functional roles onto specific regions of their structure. The architecture of the proteins appears to be modular and segments associated with voltage sensing and the pore lining have been identified. However, the means by which movement of the sensor is transduced into channel opening is still unclear. In this communication, we report on a chimeric potassium channel construct which can function in two distinct operating voltage ranges, spanning both inward and outward currents with a non-conducting intervening regime. The observed changes in operating range could be brought about by perturbing either the direction of sensor movement or the process of transducing movements of the sensor into channel opening and closing. The construct could thus provide a means to identify the machinery underlying these processes.     

Myoblast migration is prevented by a calpain-dependent accumulation of MARCKS

Calpains, also called calcium activated neutral cysteine proteases are presently known to play pivotal roles in physiological and biological phenomena such as signal transduction, cell spreading and motility, apoptosis, regulation of cell cycle and regulation of muscle cell differentiation. Concerning this last point, calpains have been shown to play a crucial role during the earlier myogenesis. In this study we have analyzed the involvement of calpains during an important step of myogenesis: myoblast migration. Our findings show that myoblast migration was drastically reduced when the expression of micro- and m-calpain was decreased. We have also observed that MARCKS (myristoylated alanine rich C kinase substrate), a protein localized at focal adhesion sites, was significantly accumulated when the expression levels of calpains were decreased. Also, using phorbol myristate acetate, (an activator of PKC) and plasmids carrying the full-length cDNA of MARCKS or a cDNA fragment lacking the phosphorylation site domain, we demonstrated that normal myoblast migration is dependent on MARCKS phosphorylation and localization.     

Myristoylation-dependent N-terminal cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cellular extracts

The myristoylated alanine-rich C kinase substrate (MARCKS) has been proposed to regulate the plasticity of the actin cytoskeleton at its site of attachment to membranes. In macrophages, MARCKS is implicated in various cellular events including motility, adhesion and phagocytosis. In this report we show that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E. coli, between Lys-6 and Thr-7. Cleavage of MARCKS decreases its affinity for macrophage membranes by ca. one order of magnitude, highlighting the contribution of the myristoyl moiety of MARCKS to membrane binding. Importantly, cleavage requires myristoylation of MARCKS. Furthermore, MARCKS-related protein (MRP), the second member of the MARCKS family, is not digested. Since Thr-7 is lacking in MRP this suggests that Thr-7 at the P1 position is important for the recognition of lipid-modified substrates. A different product is observed when MARCKS is incubated with a calf brain cytosolic extract. This product can be remyristoylated in the presence of myristoyl-CoA and N-myristoyl transferase, demonstrating that cycles of myristoylation/demyristoylation of MARCKS can be achieved in vitro. Although the physiological relevance of these enzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrates.     

Lipotropha dorci n. sp. [Neogregarinida], parasite des larves deDorcus parallelipipedus L. [Coleopt. scarab.]

Résumé Des larves deDorcus parallelipipedus L. provenant de Saint-Hilaire-de-Brethmas (Gard) dans la région méditerranéenne de France et récoltées dans des troncs décomposés de chataigniers, se sont révélées intensément parasitées par des Grégarines appartenant à l'ordre desNeogregarinida. Nous étudions leur localisation dans le tissu adipeux, leur cycle, l'action pathogène sur l'h?te et discutons leur place systématique.

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Summary Lipotropha dorci n. sp. (Neogregarinida) is a parasite developing inDorcus parallelepipedus. The parasite is situated only in the fat body which can be destroyed completely by its activity. The life cycle ofLipotropha includes only one type of schizogony with 3–4 μ long merozo?ts, the spherical uninucleate gametocytes are gathered in pairs and form 23 μ long gametokysts. These give rise to 11 to 12 μ long sporocysts with a thick, non ornamented envelope, gathered round rests of cytoplasma. The size of these different stages enables to distinguishLipotropha dorci from the already described species:Lipotropha macrospora Keilin andLipotropha microspora Keilin.