Identification of pheromone-like compounds in male reproductive organs of the oriental locust Locusta migratoria

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.     

Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system?

The present study reports the results of a morpho-functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin-synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosinehydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p-phenylenediamine-pyrocatechol (PPD-PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenoloxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue-specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Gene cloning and protein expression of γ-glutamyltranspeptidases from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>: comparison of molecular and structural properties with mesophilic counterparts

γ-Glutamyltranspeptidase (γ-GT) is an ubiquitous enzyme that catalyzes the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. γ-GTs from extremophiles, bacteria adapted to live in hostile environments, were selected as model systems to study the molecular underpinnings of their adaptation to extreme conditions and to find out special properties of potential biotechnological interest. Here, we report the cloning, expression and purification of two members of γ-GT family from two different extremophilic species, Thermus thermophilus (TtGT) and Deinococcus radiodurans (DrGT); the first is an aerobic eubacterium, growing at high temperatures (50–82°C), the second is a polyextremophile, as it tolerates radiations, cold, dehydration, vacuum, and acid. TtGT and DrGT were both synthesized as precursor proteins of 59–60 kDa, undergoing an intramolecular auto-cleavage to yield two subunits of 40 and 19–20 kDa, respectively. However, like the γ-GT from Geobacillus thermodenitrificans, but differently from the other characterized bacterial and eukaryotic γ-GTs, the two new extremophilic enzymes displayed γ-glutamyl hydrolase, but not transpeptidase activity in the 37–50°C temperature range, pH 8.0. The comparison of sequences and structural models of these two proteins with experimental-determined structures of other known mesophilic γ-GTs suggests that the extremophilic members of this protein family have found a common strategy to adapt to different hostile environments. Moreover, a phylogenetic analysis suggests that γ-GTs displaying only γ-glutamyl hydrolase activity could represent the progenitors of the bacterial and eukaryotic counterparts.     

The magic spot ppGpp influences in vitro the molecular and functional properties of the elongation factor 1α from the archaeon Sulfolobus solfataricus

Guanosine tetra-phosphate (ppGpp), also known as magic spot I, is a key molecule in the stringent control of most eubacteria and some eukarya. Here, we show that ppGpp affects the functional and molecular properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α). Indeed, ppGpp inhibited archaeal protein synthesis in vitro, even though the concentration required to get inhibition was higher than that required for the eubacterial and eukaryal systems. Regarding the partial reactions catalysed by SsEF-1α the effect produced by ppGpp on the affinity for aa-tRNA was lower than that measured in the presence of GTP but higher than that for GDP. Magic spot I was also able to bind SsEF-1α with an intermediate affinity in comparison to that displayed by GDP and GTP. Furthermore, ppGpp inhibited the intrinsic GTPase of SsEF-1α with a competitive behaviour. Finally, the binding of ppGpp to SsEF-1α rendered the elongation factor more resistant to heat treatment and the analysis of the molecular model of the complex between SsEF-1α and ppGpp suggests that this stabilisation arises from the charge optimisation on the surface of the protein.     

A role for PKCε during C2C12 myogenic differentiation

In a previous report we have demonstrated that PLCγ1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCγ1-dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCε and η expression, and that PKCε is able to form a complex with PLCγ1. The increase in PKCε amount during myogenic differentiation is associated to an increase in PKCε activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCγ1 and PKCε localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCγ1 and PKCε increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCγ1–PKCε complex co-localizes with protein 58 K, a specific Golgi marker. Moreover, our results indicate that the Golgi-associated PKCε form, i.e. PKCε phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCγ1, we analyzed the effect on cyclin D3 expression of either PKCε overexpression or silencing, in order to investigate whether PKCε could also affect cyclin D3 expression. The results showed that either a modification of PKCε expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCε in regulating insulin inositide-dependent PLCγ1 signalling in skeletal muscle differentiation.     

Ofd1 Controls Dorso-Ventral Patterning and Axoneme Elongation during Embryonic Brain Development

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.     

Structural and functional insights into IκB-α/HIV-1 Tat interaction

Protein-protein interactions play fundamental roles in physiological and pathological biological processes. The characterization of the structural determinants of protein-protein recognition represents an important step for the development of molecular entities able to modulate these interactions. We have recently found that IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) blocks the HIV-1 expression and replication in a NF-κB-independent manner by directly binding to the virus-encoded Tat transactivator. Here, we report the evaluation of the entity of binding of IκB-α to Tat through in vitro Surface Plasmon Resonance assay. Moreover, by designing and characterizing a set of peptides of the C-terminus region of IκB-α, we show that the peptide corresponding to the IκB-α sequence 262-287 was able to bind to Tat with high affinity (300 nM). The characterization of a number of IκB-α-based peptides also provided insights into their intrinsic folding properties. These findings have been corroborated by mutagenesis studies on the full-length IκB-α, which unveil that different IκB-α residues are involved in NF-κB or Tat recognition.     

Molecular and functional properties of the psychrophilic elongation factor G from the Antarctic Eubacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC 125

The molecular and functional properties of the elongation factor (EF) G from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) were studied. PhEF-G catalyzed protein synthesis in vitro that was inhibited by fusidic acid, an antibiotic specifically acting on EF-G. The EF interacted with GDP only in the presence of P. haloplanktis ribosome and fusidic acid with an affinity similar to that displayed by Escherichia coli EF-G. The psychrophilic translocase elicited a ribosome-dependent GTPase that was competitively inhibited by GDP, the slowly hydrolyzable GTP analog GppNHp, and the protein synthesis inhibitor ppGDP. The temperature dependence of the activity of PhEF-G reached its maximum at least 26°C beyond the growth temperature of P. haloplanktis (4–20°C). The heat inactivation profile of the ribosome-dependent GTPase of PhEF-G gave a temperature for half inactivation (46°C), significantly lower than that for half denaturation measured by either UV- (57°C) or fluorescence-melting (62°C). This finding was attributed to a different effect of the temperature on the catalytic domain with respect to that elicited on the other domains constituting the EF, thus confirming the differential molecular flexibility present in psychrophilic enzymes. A molecular model, based on the 3D coordinates of a thermophilic EF-G, showed differences only in connecting loops. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.