Gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.

A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein¿Raf1 and the Raichu-KRas probe, we identified for the first time in vivo¿active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14¿MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell.

E2F1 regulates cellular growth by mTOR signaling

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.

Phenotypic and molecular assessment of seven patients with 6p25 deletion syndrome: relevance to ocular dysgenesis and hearing impairment.

BACKGROUND: Thirty-nine patients have been described with deletions involving chromosome 6p25. However, relatively few of these deletions have had molecular characterization. Common phenotypes of 6p25 deletion syndrome patients include hydrocephalus, hearing loss, and ocular, craniofacial, skeletal, cardiac, and renal malformations. Molecular characterization of deletions can identify genes that are responsible for these phenotypes. METHODS: We report the clinical phenotype of seven patients with terminal deletions of chromosome 6p25 and compare them to previously reported patients. Molecular characterization of the deletions was performed using polymorphic marker analysis to determine the extents of the deletions in these seven 6p25 deletion syndrome patients. RESULTS: Our results, and previous data, show that ocular dysgenesis and hearing impairment are the two most highly penetrant phenotypes of the 6p25 deletion syndrome. While deletion of the forkhead box C1 gene (FOXC1) probably underlies the ocular dysgenesis, no gene in this region is known to be involved in hearing impairment. CONCLUSIONS: Ocular dysgenesis and hearing impairment are the two most common phenotypes of 6p25 deletion syndrome. We conclude that a locus for dominant hearing loss is present at 6p25 and that this locus is restricted to a region distal to D6S1617. Molecular characterization of more 6p25 deletion patients will aid in refinement of this locus and the identification of a gene involved in dominant hearing loss.

Biomass production and phosphorus use of forage grasses fertilized with two phosphorus sources

A major constraint to agricultural production in acid soils of tropical regions is the low soil P availability, due to the high adsorption capacity, low P level in the source material and low efficiency of P uptake and use by most of the modern varieties grown commercially. This study was carried out to evaluate the biomass production and P use by forage grasses on two soils fertilized with two P sources of different solubility. Two experiments were carried out, one for each soil (Cambisol and Latosol), using pots filled with 4 dm³ soil in a completely randomized design and a 4 x 2 factorial scheme. The treatments consisted of a combination of four forage plants (Brachiaria decumbens, Brachiaria brizantha, Pennisetum glaucum and Sorghum bicolor) with two P sources (Triple Superphosphate - TSP and Arad Reactive Phosphate - ARP), with four replications. The forage grasses were harvested at pre-flowering, when dry matter weight and P concentrations were measured. Based on the P concentration and dry matter production, the total P accumulation was calculated. With these data, the following indices were calculated: the P uptake efficiency of roots, P use efficiency, use efficiency of available P, use efficiency of applied P and agronomic efficiency. The use of the source with higher solubility (TSP) resulted, generally, in higher total dry matter and total P accumulation in the forage grasses, in both soils. For the less reactive source (ARP), the means found in the forage grasses, for use efficiency and efficient use of available P, were always higher when grown in Latosol, indicating favorable conditions for the solubility of ARP. The total dry matter of Brachiaria brizantha was generally higher, with low P uptake, accumulation and translocation, which indicated good P use efficiency for both P sources and soils. The forage plants differed in the P use potential, due to the sources of the applied P and of the soils used. Less than 10 % of the applied P was immobilized in the forage dry matter. Highest values were observed for TSP, but this was not reflected in a higher use efficiency of P from this source.

Phloem mobility of Boron in two eucalypt clones

Boron deficiency causes large productivity losses in eucalypt stands in extensive areas of the Brazilian Cerrado region, thus understanding B mobility is a key step in selecting genetic materials that will better withstand B limitation. Thus, in this study B mobility was evaluated in two eucalypt clones (68 and 129), under B sufficiency or B deficiency, after foliar application of the 10B isotope tracer to a single mature leaf. Samples of young tissue, mature leaves and roots were collected 0, 1, 5, 12 and 17 days after 10B application. The 10B:11B isotope ratio was determined by HR-ICP-MS. Samples of leaves and xylem sap were collected for the determination of soluble sugars and polyalcohols by ion chromatography. Boron was translocated within eucalypt. Translocation of foliar-applied 10B to the young tissues, mature leaves and roots was higher in clone 129 than in 68. Seventeen days after 10B application to a single mature leaf, between 14 and 18 % of B in the young tissue was originated from foliar B application. In plants with adequate B supply the element was not translocated out of the labeled leaf.

Boron mobility in eucalyptus clones

Understanding the magnitude of B mobility in eucalyptus may help to select clones that are more efficient for B use and to design new practices of B fertilization. This study consisted of five experiments with three eucalyptus clones (129, 57 and 58) where the response to and mobility of B were evaluated. Results indicated that clone 129 was less sensitive to B deficiency than clones 68 and 57, apparently due to its ability to translocate B previously absorbed via root systems to younger tissues when B in solution became limiting. Translocation also occurred when B was applied as boric acid only once to a single mature leaf, resulting in higher B concentration in roots, stems and younger leaves. The growth of B-deficient plants was also recovere by a single foliar application of B to a mature leaf. This mobility was greater, when foliar-applied B was supplied in complexed (boric acid + manitol) than in non-complexed form (boric acid alone). When the root system of clone 129 was split in two solution compartments, B supplied to one root compartment was translocated to the shoot and back to the roots in the other compartment, improving the B status and growth. Thus, it appears that B is relatively mobile in eucalyptus, especially in clone 129, and its higher mobility could be due to the presence of an organic compound such as manitol, able to complex B.

Genome-wide arrays in routine diagnostics of hematological malignancies.

Over the last three decades, cytogenetic analysis of malignancies has become an integral part of disease evaluation and prediction of prognosis or responsiveness to therapy. In most diagnostic laboratories, conventional karyotyping, in conjunction with targeted fluorescence in situ hybridization analysis, is routinely performed to detect recurrent aberrations with prognostic implications. However, the genetic complexity of cancer cells requires a sensitive genome-wide analysis, enabling the detection of small genomic changes in a mixed cell population, as well as of regions of homozygosity. The advent of comprehensive high-resolution genomic tools, such as molecular karyotyping using comparative genomic hybridization or single-nucleotide polymorphism microarrays, has overcome many of the limitations of traditional cytogenetic techniques and has been used to study complex genomic lesions in, for example, leukemia. The clinical impact of the genomic copy-number and copy-neutral alterations identified by microarray technologies is growing rapidly and genome-wide array analysis is evolving into a diagnostic tool, to better identify high-risk patients and predict patients' outcomes from their genomic profiles. Here, we review the added clinical value of an array-based genome-wide screen in leukemia, and discuss the technical challenges and an interpretation workflow in applying arrays in the acquired cytogenetic diagnostic setting.

One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription.

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK- phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.

Potassium availability in a hapludalf soil under long term fertilization

In a system in which fertilization is recommended, diagnosis of soil K availability and the establishment of critical levels are made difficult by the possibility of a contribution of non-exchangeable forms of K for plant nutrition. Due to its magnitude, this contribution is well diagnosed in long term experiments and in those which compare fertilization systems with positive and negative balances in terms of replacement of the K extracted by plants. The objective of this study was to evaluate K availability in a Hapludalf under fertilization for sixteen years with the addition of K doses. The study was undertaken in an experiment set up in 1991 and carried out until 2007 in the experimental area of the Soil Department of the Federal University of Santa Maria (Universidade Federal de Santa Maria - UFSM), in Santa Maria (RS), Brazil. The soil was a Typic Hapludalf submitted to four doses of K (0, 60, 120 and 180 kg ha-1 K2O) and subdivided in the second year, when 60 kg ha-1 of K2O were reapplied in the subplots in 0, 1, 2 and 3 times. As of the fifth year, the procedure was repeated. Grain yield above ground dry matter and total K content contained in the plant tissue were evaluated. Soil samples were collected, oven dried, ground, passed through a sieve and submitted to exchangeable K analysis by the Mehlich-1 extractor; non-exchangeable K by boiling HNO3 1 mol L-1 and total K by HF digestion. Potassium fertilization guidelines should foresee the establishment of a critical level as of which the recommended dose should accompany crop needs, which coincides with the quantity exported by the grain, without there being the need for the creation of broad ranges of K availability to predict K fertilization. In adopting the K fertilization recommendations proposed in this manner, there will not be K translocation in the soil profile.

Growth of seedlings of pigeon pea (Cajanus cajan (l.) millsp), wand riverhemp (Sesbania virgata (cav.) pers.), and lead tree (Leucaena leucocephala (lam.) de wit) in an arsenic-contaminated soil

Phytoremediation strategies utilize plants to decontaminate or immobilize soil pollutants. Among soil pollutants, metalloid As is considered a primary concern as a toxic element to organisms. Arsenic concentrations in the soil result from anthropogenic activities such as: the use of pesticides (herbicides and fungicides); some fertilizers; Au, Pb, Cu and Ni mining; Fe and steel production; coal combustion; and as a bi-product during natural gas extraction. This study evaluated the potential of pigeon pea (Cajanus cajan), wand riverhemp (Sesbania virgata), and lead tree (Leucaena leucocephala) as phytoremediators of soils polluted by As. Soil samples were placed in plastic pots, incubated with different As doses (0; 50; 100 and 200 mg dm-3) and then sown with seeds of the three species. Thirty (pigeon pea) and 90 days after sowing, the plants were evaluated for height, collar diameter and dry matter of young, intermediate and basal leaves, stems and roots. Arsenic concentration was determined in different aged leaves, stems and roots to establish the translocation index (TI) between the plant root system and aerial plant components and the bioconcentration factors (BF). The evaluated species showed distinct characteristics regarding As tolerance, since the lead tree and wand riverhemp were significantly more tolerant than pigeon pea. The high As levels found in wand riverhemp roots suggest the existence of an efficient accumulation and compartmentalization mechanism in order to reduce As translocation to shoot tissues. Pigeon pea is a sensitive species and could serve as a potential bioindicator plant, whereas the other two species have potential for phytoremediation programs in As polluted areas. However, further studies are needed with longer exposure times in actual field conditions to reach definite conclusions on relative phytoremediation potentials.

Bax-induced apoptosis in Leber's congenital amaurosis : a dual role in rod and cone degeneration

Purpose:We previously observed that anti- and pro-apoptotic genes of the Bcl-2 family were differentially expressed during the development of LCA in the Rpe65-/- mouse model (Cottet et al. 2006). Moreover, we reported that activation and translocation of pro-apoptotic Bax to the mitochondria was associated with apoptosis of rod photoreceptors as the disease progressed (Cottet et al. 2008). In this study we challenged whether disruption of the pro-apoptotic pro-apoptotic Bax protein is sufficient to protect photoreceptor cells against apoptosis. Methods:Apoptosis of photoreceptor cells was addressed by TUNEL assay on flatmounted retinas. Counting of the rod nuclei within the ONL was performed following hematoxylin/eosin histological staining of retina sections. Expression level and localization of photoreceptor gene markers were assessed by quantitative PCR and immunohistological analyses. Results:While expression of rod photoreceptor genes was decreased in Rpe65-deficient retina, expression level remained unchanged in Rpe65-/- / Bax-/- mice. Moreover, OS dysorganization and shortening as well as decrease in ONL thickness observed in diseased retina were prevented in mice lacking functional Bax protein. TUNEL assay confirmed that Bax-dependent rod photoreceptor apoptosis was abolished in Rpe65-/- / Bax-/- mice. However, early and fast degeneration of cone cells was not prevented in Rpe65-/- / Bax-/- mice, indicating that Bax-induced apoptotic pathway was not involved in the degenerating process of cones in Rpe65-deficient retina. Conclusions:Altogether, these data show for the first time that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in LCA disease. While pro-apoptotic Bax is essential to trigger rod photoreceptor apoptosis, early degeneration of cones is not dependent on Bax-mediated apoptotic pathway in Rpe65-deficientmice.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Systemic mastocytosis with associated myeloproliferative disease and precursor B lymphoblastic leukaemia with t(13;13)(q12;q22) involving FLT3.

Systemic mastocytoses represent neoplastic proliferations of mast cells. In about 20% of cases systemic mastocytoses are accompanied by clonal haematopoietic non-mast cell-lineage disorders, most commonly myeloid neoplasms. A case of systemic mastocytosis carrying the characteristic mutation at codon 816 (D816V) in the KIT gene of mast cells, with two concurrent accompanying clonal haematopoietic non-mast cell-lineage disorders, chronic myeloproliferative disease, unclassifiable and precursor B lymphoblastic leukaemia is documented. Both accompanying clonal haematopoietic non-mast cell-lineage disorders carried the wild-type KIT gene, but had a novel t(13;13)(q12;q22) involving the FLT3 locus at 13q12. The chronic myeloproliferative disease, unclassifiable and the precursor B lymphoblastic leukaemia were cured by syngenous stem cell transplantation, but the systemic mastocytosis persisted for more than 10 years. The additional impact of molecular techniques on the correct diagnosis in haematological malignancies is highlighted, and evidence is provided that, apart from internal tandem duplications and mutations, FLT3 can be activated by translocations.

Insulin and insulin-like growth factor I receptors utilize different G protein signaling components.

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.