The primate-specific protein TBC1D3 is required for optimal macropinocytosis in a novel ARF6-dependent pathway

The generation of novel genes and proteins throughout evolution has been proposed to occur as a result of whole genome and gene duplications, exon shuffling, and retrotransposition events. The analysis of such genes might thus shed light into the functional complexity associated with highly evolved species. One such case is represented by TBC1D3, a primate-specific gene, harboring a TBC domain. Because TBC domains encode Rab-specific GAP activities, TBC-containing proteins are predicted to play a major role in endocytosis and intracellular traffic. Here, we show that the TBC1D3 gene originated late in evolution, likely through a duplication of the RNTRE locus, and underwent gene amplification during primate speciation. Despite possessing a TBC domain, TBC1D3 is apparently devoid of Rab-GAP activity. However, TBC1D3 regulates the optimal rate of epidermal growth factor–mediated macropinocytosis by participating in a novel pathway involving ARF6 and RAB5. In addition, TBC1D3 binds and colocalize to GGA3, an ARF6-effector, in an ARF6-dependent manner, and synergize with it in promoting macropinocytosis, suggesting that the two proteins act together in this process. Accordingly, GGA3 siRNA-mediated ablation impaired TBC1D3-induced macropinocytosis. We thus uncover a novel signaling pathway that appeared after primate speciation. Within this pathway, a TBC1D3:GGA3 complex contributes to optimal propagation of signals, ultimately facilitating the macropinocytic process.     

Comparison between kinematic and kinetic methods for computing the vertical displacement of the center of mass during human hopping at different frequencies

Kinematic and kinetic methods (sacral marker, reconstructed pelvis, segmental analysis, and force platform methods) have been used to calculate the vertical excursion of the center of mass (COM) during movement. In this study we compared the measurement of vertical COM displacement yielded by different methods during able-bodied subjects' hopping at different frequencies (varying between 1.2 and 3.2 Hz). ANOVA revealed a significant interaction between hopping frequency and method (p < 0.001), showing that increasing hopping frequency reduced the differences between methods. A post hoc analysis revealed a significant difference between all methods at the lowest hopping frequency and between the force platform and both the sacral marker and reconstructed pelvis methods at the intermediate hopping frequencies, with differences ranging from 16 to 67 millimeters (all p < 0.05). Results are discussed in view of each methods' limits. We conclude that the segmental analysis and force platform methods can be considered to provide the most accurate results for COM vertical excursion during human hopping in a large range of hopping frequency.     

Computational studies of the structure, dynamics and native content of amyloid-like fibrils of ribonuclease A

It is a common belief that some residues of a protein are more important than others. In some cases, point mutations of some residues make butterfly effect on the protein structure and function, but in other cases they do not. In addition, the residues important for the protein function tend to be not only conserved but also coevolved with other interacting residues in a protein. Motivated by these observations, the authors propose that there is a network composed of the residues, the residue-residue coevolution network (RRCN), where nodes are residues and links are set when the coevolutionary interaction strengths between residues are sufficiently large. The authors build the RRCN for the 44 diverse protein families. The interaction strengths are calculated by using McBASC algorithm. After constructing the RRCN, the authors identify residues that have high degree of connectivity (hub nodes), and residues that play a central role in network flow of information (C(I) nodes). The authors show that these residues are likely to be functionally important residues. Moreover, the C(I) nodes appear to be more relevant to the function than the hub nodes. Unlike other similar methods, the method described in this study is solely based on sequences. Therefore, the method can be applied to the function annotation of a wider range of proteins.     

Biodiversity of yeasts isolated from the indigenous forest of Argan <Emphasis Type="Italic">(Argania spinosa</Emphasis> (L.) Skeels) in Morocco

In this study we have isolated and characterized yeasts from the soil, leaves and fruits of the indigenous Moroccan Argan tree (Argania spinosa) in two locations: the coastal city of Essaouira and a drier, more stressed environment in Taroudant city. Factorial and classification analyses of the metabolic profiles showed that the yeasts from the soil and those from the fruit seemed to form distinctive groups while those from the leaves were common to the two groups. Associating the profiles with yeast species, the soil isolates seemed to be dominated by profiles associated with basidiomycetous yeasts (Bullera variabilis, association to Filobasidium capsuligenum, and Rhodotorula glutinis) while those of the fruits were associated with ascomycetous yeasts (Pichia angusta and Zygoascus hellenicus). Most profile groups were shared between the leaves and one of the other biotopes owing to the semi-deciduous character of the Argan leaves that dominate in the rhizospheric soil and to the fibrous and low flesh fruits of Argan. Although most metabolic profile groups were represented in both sampling locations, certain groups were encountered only in Taroudant samples among which a group of four yeasts that grew at 44 °C. The Taroudant samples also presented the two most osmo-tolerant yeasts capable of growing at 15% NaCl and 125% sucrose. Some of the yeast strains showed very promising activities of polygalacturonase (0.40 units/g protein) without any pectinesterase activity while others strongly inhibited the gray rot mould Botrytis cinerea, and could be good candidates for the post-harvest control of this mould on fruits.     

Pathophysiology of the human intervertebral disc

Intervertebral disc degeneration is a common invalidating disorder that can affect the musculoskeletal apparatus in both younger and older ages. The chief component of the intervertebral disc is the highly organized extracellular matrix; maintenance of its organization is essential for correct spinal mechanics. The matrix components, mainly proteoglycans and collagens, undergo a slow and continuous cell-mediated turnover process that enables disc cells to adapt their environment to external stimuli. Cellular senescence and a history of chronic abnormal loading can upset this balance, leading to progressive tissue failure that results in disc degeneration. Although biological treatment approaches to disc repair are still far to come, advances in our understanding of disc biochemistry and in defining the role of genetic inheritance have provided a starting point for developing new concepts in the diagnosis, therapy and prevention of disc degeneration.     

Archaeal protoglobin structure indicates new ligand diffusion paths and modulation of haem-reactivity

The structural adaptability of the globin fold has been highlighted by the recent discovery of the 2-on-2 haemoglobins, of neuroglobin and cytoglobin. Protoglobin from Methanosarcina acetivorans C2A-a strictly anaerobic methanogenic Archaea-is, to the best of our knowledge, the latest entry adding new variability and functional complexity to the haemoglobin (Hb) superfamily. Here, we report the 1.3 A crystal structure of oxygenated M. acetivorans protoglobin, together with the first insight into its ligand-binding properties. We show that, contrary to all known globins, protoglobin-specific loops and an amino-terminal extension completely bury the haem within the protein matrix. Access of O(2), CO and NO to the haem is granted by the protoglobin-specific apolar tunnels reaching the haem distal site from locations at the B/G and B/E helix interfaces. Functionally, M. acetivorans dimeric protoglobin shows a selectivity ratio for O(2)/CO binding to the haem that favours O(2) ligation and anticooperativity in ligand binding. Both properties are exceptional within the Hb superfamily.     

Binding of recombinant PrPc to human plasminogen: kinetic and thermodynamic study using a resonant mirror biosensor

Transmissible spongiform encephalopathies are a class of sporadic, genetic and transmissible neurodegenerative diseases that affect both humans and animals. Propagation of these diseases is thought to be due to the misfolding of a neuronal glyco-protein, PrP(c), into a pathological insoluble conformer, PrP(Sc). In earlier works, some serum components were identified as exclusive PrP(Sc)-interacting proteins (Fisher et al., Nature 2000;408:479), and thus those macromolecules were thought to represent a potential diagnostic endogenous factor discriminating between normal and pathological prion proteins. In contrast, in agreement with a recent work (Kornblatt et al., Biochem Biophys Res Commun 2003;305:518), in this paper we present a detailed thermodynamic and kinetic characterization of the interaction between recombinant bovine PrP(c 25-242) and the human serum component plasminogen, measured using a resonant mirror technique: our results reveal a high-affinity interaction between the two binding partners. For comparison, the complex obtained from the purified full-length PrP(c) and human plasminogen was also studied: both prion proteins (the recombinant bovine PrP(c 25-242) and the purified full-length PrP(c)) are able to bind human plasminogen. Both kinetic and thermodynamic parameters are affected by the modulation exerted by the H(+) ions in solution. Moreover, the analysis of binding, according to canonical linkage relationships, suggests the involvement of a His residue, consistent with the interaction between other serine (pro)enzymes and their ligands.     

Antioxidant status (CoQ10 and Vit. E levels) and immunohistochemical analysis of soft tissues in periodontal diseases

Reactive oxygen species and antioxidant status in periodontal diseases and periodontal-related pathologies is an item of growing interest. Immunohistochemical approach may be usefully employed in the study of soft tissues affected by periodontal disease, giving valuable information on tissue morphology and vascular proliferation that depends directly on the inflammatory state. In order to study CoQ(10) and vitamin E content in healthy gingiva and in gingivitis a new adaptation to previously published methods for their determination was adopted. During gingivitis tissue displayed a large inflammatory infiltration in the lamina propria and a VEGF positive squamous epithelium. The inflammatory infiltration consisted mainly of lymphocytes, plasma cells and neutrophils. Vitamin E dramatically decreased and CoQ(10) remained unchanged despite the increased amount of cells present in the periodontally affected tissues, indicating that continuous oxidative stress which occurred in these structure affected the antioxidant pattern of the tissue.     

Gene CATCHR--gene cloning and tagging for Caenorhabditis elegans using yeast homologous recombination: a novel approach for the analysis of gene expression

Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression.