Paper de la quinasa IKKalpha al càncer colorectal

Prèviament havíem identificat la presència d’una forma de l’IKKα de 45kD que s’expressa específicament en el nucli de les cèl.lules de cancer colorectal. A més havíem demostrat que aquesta forma estava fosforilada la qual cosa suggereix que es tracta d’una forma activa d’IKKa. Un dels objectius que ens varem plantejar va ser determinar el mecanisme per el qual es generava aquesta forma de la quinasa. Mitjançant eines bioinformàtiques hem identificat possibles llocs de processament proteolític en la seqüència d’IKKα que podrien ser responsables de generar el fragment de 45kD present en les cèl.lules tumorals. Després, hem demostrat que les proteases identificades in silico són capaces de processar IKKa in vitro, específicament en els llocs predits. S’ha pogut constatar, mitjançant la mutació dels possibles llocs de processament, que només un dels llocs identificats bioinformàticament corresponent a Cathepsin B/L era funcional in vitro, mentre que els altres llocs predits no ho eren. D’igual manera, l’expressió ectòpica de la Cathepsin B o L és capaç de produïr el processament d’IKKα. Pel contrari, la inhibició de l’activitat de la proteasa mitjançant inhibidors específics és capaç de bloquejar el processament d’IKKa en cèl.lules tumorals. Finalment, hem demostrat que els nivells de la Cathepsin B i L, proteases identificades com a responsables de processar IKKa es troben sobre-expressades en la majoria de les mostres humanes de càncer de colon analitzades comparat amb el teixit normal adjacent del mateixos pacients.

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.

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.

Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics

Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis.

Angiogenesis is an important process in chronic inflammatory diseases. We observed that sera from patients with systemic vasculitis stimulated angiogenesis in an in vitro model using human umbilical vein endothelial cells cultured on a basement membrane (Matrigel) substrate. After 40% ammonium sulfate precipitation, angiogenic activity remained in the low molecular weight fraction and could be inactivated by heat. SDS-page of serum FPLC fractions exhibiting maximal angiogenic activity demonstrated two prominent species of 45 and 16-20 kD in patients' sera. These bands were much less apparent in sera obtained from control subjects. Amino-terminal sequencing of the 45-kD protein demonstrated that it was haptoglobin. Purified haptoglobin stimulated angiogenesis in a dose-dependent manner. The angiogenic activity of vasculitis patients' sera was partially inhibited by an antihaptoglobin antibody. Furthermore, serum haptoglobin levels in vasculitis patients correlated both with disease and angiogenic activity. Haptoglobin angiogenic activity was confirmed in two in vivo models using an implanted disc and a subcutaneous injection of basement membrane. Stimulation of angiogenesis is a newly recognized biological function of haptoglobin. The increased levels of haptoglobin found in chronic inflammatory conditions may play an important role in tissue repair. In systemic vasculitis, haptoglobin might also compensate for ischemia by promoting development of collateral vessels.

Study and modeling of the desintegration kinetics of coated paper

The disintegration of recovered paper is the first operation in the preparation of recycled pulp. It is known that the defibering process follows a first order kinetics from which it is possible to obtain the disintegration kinetic constant (KD) by means of different ways. The disintegration constant can be obtained from the Somerville index results (%lsv and from the dissipated energy per volume unit (Ss). The %slv is related to the quantity of non-defibrated paper, as a measure of the non-disintegrated fiber residual (percentage of flakes), which is expressed in disintegration time units. In this work, disintegration kinetics from recycled coated paper has been evaluated, working at 20 revise rotor speed and for different fiber consistency (6, 8, 10, 12 and 14%). The results showed that the values of experimental disintegration kinetic constant, Ko, through the analysis of Somerville index, as function of time. Increased, the disintegration time was drastically reduced. The calculation of the disintegration kinetic constant (modelled Ko), extracted from the Rayleigh’s dissipation function, showed a good correlation with the experimental values using the evolution of the Somerville index or with the dissipated energy

Study and modeling of the desintegration kinetics of coated paper

The disintegration of recovered paper is the first operation in the preparation of recycled pulp. It is known that the defibering process follows a first order kinetics from which it is possible to obtain the disintegration kinetic constant (KD) by means of different ways. The disintegration constant can be obtained from the Somerville index results (%lsv and from the dissipated energy per volume unit (Ss). The %slv is related to the quantity of non-defibrated paper, as a measure of the non-disintegrated fiber residual (percentage of flakes), which is expressed in disintegration time units. In this work, disintegration kinetics from recycled coated paper has been evaluated, working at 20 revise rotor speed and for different fiber consistency (6, 8, 10, 12 and 14%). The results showed that the values of experimental disintegration kinetic constant, Ko, through the analysis of Somerville index, as function of time. Increased, the disintegration time was drastically reduced. The calculation of the disintegration kinetic constant (modelled Ko), extracted from the Rayleigh’s dissipation function, showed a good correlation with the experimental values using the evolution of the Somerville index or with the dissipated energy

Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64A

A genomic clone (p268c) coding for the 28 kD storage protein Zc2 from maize endosperm has been isolated and sequenced.

Sequence analysis of a genomic clone encoding a Zc2 protein from Zea mays W64A

A genomic clone (p268c) coding for the 28 kD storage protein Zc2 from maize endosperm has been isolated and sequenced.

Study and modeling of the desintegration kinetics of coated paper

The disintegration of recovered paper is the first operation in the preparation of recycled pulp. It is known that the defibering process follows a first order kinetics from which it is possible to obtain the disintegration kinetic constant (KD) by means of different ways. The disintegration constant can be obtained from the Somerville index results (%lsv and from the dissipated energy per volume unit (Ss). The %slv is related to the quantity of non-defibrated paper, as a measure of the non-disintegrated fiber residual (percentage of flakes), which is expressed in disintegration time units. In this work, disintegration kinetics from recycled coated paper has been evaluated, working at 20 revise rotor speed and for different fiber consistency (6, 8, 10, 12 and 14%). The results showed that the values of experimental disintegration kinetic constant, Ko, through the analysis of Somerville index, as function of time. Increased, the disintegration time was drastically reduced. The calculation of the disintegration kinetic constant (modelled Ko), extracted from the Rayleigh’s dissipation function, showed a good correlation with the experimental values using the evolution of the Somerville index or with the dissipated energy

Study and modeling of the desintegration kinetics of coated paper

The disintegration of recovered paper is the first operation in the preparation of recycled pulp. It is known that the defibering process follows a first order kinetics from which it is possible to obtain the disintegration kinetic constant (KD) by means of different ways. The disintegration constant can be obtained from the Somerville index results (%lsv and from the dissipated energy per volume unit (Ss). The %slv is related to the quantity of non-defibrated paper, as a measure of the non-disintegrated fiber residual (percentage of flakes), which is expressed in disintegration time units. In this work, disintegration kinetics from recycled coated paper has been evaluated, working at 20 revise rotor speed and for different fiber consistency (6, 8, 10, 12 and 14%). The results showed that the values of experimental disintegration kinetic constant, Ko, through the analysis of Somerville index, as function of time. Increased, the disintegration time was drastically reduced. The calculation of the disintegration kinetic constant (modelled Ko), extracted from the Rayleigh’s dissipation function, showed a good correlation with the experimental values using the evolution of the Somerville index or with the dissipated energy