The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors

GIT proteins are GTPase-activating proteins (GAPs) for ADP-ribosylation factor (ARF) small GTP-binding proteins, and interact with the PIX family of Rac1/Cdc42 guanine nucleotide exchange factors. GIT and PIX transiently localize p21-activated protein kinases (PAKs) to remodeling focal adhesions through binding to paxillin. To understand the role of these interactions, the association of GIT and PIX proteins was examined in detail. Two separable binding interactions link GIT and PIX proteins, GIT and PIX proteins each dimerize and a beta-PIX fragment containing the GIT-binding region failed to inhibit the association of the GIT and PIX proteins. Endogenous GIT and PIX co-fractionate at a very high molecular size. Purified 6xHis-tagged beta-PIX from Sf9 cells co-expressing untagged GIT1 yields recombinant GIT1/beta-PIX complexes that have equal amounts of beta-PIX and GIT1 and co-fractionate at the same large size as native GIT/PIX complexes. Thus, GIT and PIX proteins are tightly associated as a multimeric nexus capable of linking together important signaling molecules, including PAKs.     

Abortive infection of Lutzomyia longipalpis insect vectors by aflagellated LdARL-3A-Q70L overexpressing Leishmania amazonensis parasites

Leishmania donovani ADP-ribosylation factor-like protein 3A (LdARL-3A) is a small G protein isolated from the protozoan parasite L. donovani with no defined physiological function. Previously [Cuvillier, A., Redon, F., Antoine, J.-C., Chardin, P., DeVos, T., and Merlin, G. (2000) J Cell Sci 113: 2065-2074] we have shown that overexpression in L. amazonensis promastigotes of the mutated protein LdARL-3A-Q70L, which remains constitutively associated with GTP, leads to the disappearance of the flagellum but does not impair cell viability or growth. Here we report that parasites overexpressing LdARL-3A-Q70L can invade in vitro cultivated macrophages to the same extent as control cells, demonstrating that the flagellum is not necessary for attachment to or engulfment into macrophages. These infections are productive because amastigotes differentiate and multiply. However, aflagellated LdARL-3A-Q70L-overexpressing Leishmania promastigotes could not survive in experimentally infected Lutzomyia longipalpis insect vectors, in contrast to untransfected or native LdARL-3A-overexpressing cells. Overexpression of the native and mutated proteins did not modify in vitro procyclic to metacyclic lipophosphoglycan maturation or differentiation from procyclic to metacyclic promastigotes, nevertheless there is a block in transmission of Leishmania. Better understanding of LdARL-3A pathways, notably those regarding flagellum biogenesis, may lead to the future development of Leishmania-specific drugs, which may stop parasite transmission in nature without affecting other species.     

Cloning of β-1,3-glucanases expressed during Cichorium somatic embryogenesis

Three different -1,3-glucanase cDNA fragments, CG1, CG2 and CG3, were obtained by RT-PCR from RNA isolated from Cichorium hybrid `474' leaf fragments cultured for 11 days under somatic embryogenesis-inducing conditions. When expressed in Escherichia coli the proteins encoded by the three cDNAs were recognized by antibodies raised against 38 kDa extracellular -1,3-glucanases studied previously (Helleboid et al., Planta 205 (1998) 56–63). The CG2 and CG3 cDNAs may represent expressed alleles of one gene because their sequences showed a very high identity (98.5%) and are only 70% identical with CG1. Southern blot analysis revealed the presence of 3–4 genes coding for -1,3-glucanases in the Cichorium genome. Expression analysis of the genes corresponding to the three clones analysed by semi-quantitative RT-PCR indicated that CG1 mRNAs were only detectable in Cichorium hybrid `474' leaf fragments from day 3 of somatic embryogenesis induction, whereas CG2-CG3 mRNAs were already present in non-induced leaf tissue of both the embryogenic hybrid `474' and a non-embryogenic genotype. The level of CG1 mRNAs was particularly high when embryogenic cells were dividing to produce embryos, and when the amount of callose deposited in cell walls surrounding embryogenic cells and young embryos decreased. These results indicate that expression of the CG1 gene is correlated to the somatic embryogenesis process and that it encodes a 38 kDa -1,3-glucanase protein that may be involved in the degradation of callose localized around embryogenic cells and young embryos. A full-length CG1 cDNA clone was obtained using 3 and 5 RACE-PCR, and its sequence revealed that it encodes a -1,3-glucanase that is equally homologous to both class III and class IV plant -1,3-glucanases.     

Comet assay: rapid processing of multiple samples

The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.     

Role of activity-dependent mechanisms in the control of dopaminergic neuron survival

Dopaminergic neurons that constitute the nigrostriatal pathway are characterized by singular electrical properties that allow them to discharge in vivo spontaneously in a spectrum of patterns ranging from pacemaker to random and bursting modes. These electrophysiological features allow dopaminergic neurons to optimize the release of dopamine in their terminal fields. However, there is emerging evidence indicating that electrical activity might also participate in the control of dopaminergic neuron survival, not only during development, but also in the adult brain, thus raising the possibility that alterations in ionic currents could contribute actively to the demise of these neurons in Parkinson disease. This review focuses on the mechanisms by which activity-dependent mechanisms might modulate dopaminergic cell survival.     

Amelioration of inflammation, angiogenesis and CTGF expression in an arthritis model by a TSP1-derived peptide treatment

OBJECTIVE: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. METHODS: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR. RESULTS: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals. CONCLUSION: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues.     

A missense mutation in TYRP1 causes the chocolate plumage color in chicken and alters melanosome structure

The chocolate plumage color in chickens is due to a sex‐linked recessive mutation, choc, which dilutes eumelanin pigmentation. Because TYRP1 is sex‐linked in chickens, and TYRP1 mutations determine brown coat color in mammals, TYRP1 appeared as the obvious candidate gene for the choc mutation. By combining gene mapping with gene capture, a complete association was identified between the chocolate phenotype and a missense mutation leading to a His214Asn change in the ZnA zinc‐binding domain of the protein. A diagnostic test confirmed complete association by screening 428 non‐chocolate chickens of various origins. This is the first TYRP1 mutation described in the chicken. Electron microscopy analysis showed that melanosomes were more numerous in feather follicles of chocolate chickens but exhibited an abnormal structure characterized by a granular content and an irregular shape. A similar altered morphology was observed on melanosomes of another TYRP1 mutant in birds, the roux mutation of the quail.     

Comparison of long‐stem and standard tube‐stock performance 5 years after planting in a rainforest edge on the Central Coast of NSW

Adoption of the long‐stem planting technique in riparian and nonriparian environments is increasing, despite few scientific studies examining whether the technique improves restoration outcomes. Here, we report on the results of an experiment that compares the performance of long‐stem and standard tube stock planted within a rainforest edge. Our results showed that planting technique had no significant effect on the growth and survival of Cheese Tree (Glochidion ferdinandi) and Scentless Rosewood (Synoum glandulosum) individuals, 5 years postplanting. Our findings suggest that long‐stem planting does not enhance tube‐stock performance of these two species, at least under the relatively benign environmental conditions of this study. We recommend that further tests of the technique should focus on habitats where inputs of physical energy are dominant (e.g. rivers, coastal foredunes) and in locations where (and when) growing conditions are particularly harsh.