Agreement Abstractions in Anonymous and Homonymous Distributed Systems Prone to Failures

The distributed computing models typically assume every process in the system has a distinct identifier (ID) or each process is programmed differently, which is named as eponymous system. In such kind of distributed systems, the unique ID is helpful to solve problems: it can be incorporated into messages to make them trackable (i.e., to or from which process they are sent) to facilitate the message transmission; several problems (leader election, consensus, etc.) can be solved without the information of network property in priori if processes have unique IDs; messages in the register of one process will not be overwritten by others process if this process announces; it is useful to break the symmetry. Hence, eponymous systems have influenced the distributed computing community significantly either in theory or in practice. However, every thing in the world has its own two sides. The unique ID also has disadvantages: it can leak information of the network(size); processes in the system have no privacy; assign unique ID is costly in bulk-production(e.g, sensors). Hence, homonymous system is appeared. If some processes share the same ID and programmed identically is called homonymous system. Furthermore, if all processes shared the same ID or have no ID is named as anonymous system. In homonymous or anonymous distributed systems, the symmetry problem (i.e., how to distinguish messages sent from which process) is the main obstacle in the design of algorithms. This thesis is aimed to propose different symmetry break methods (e.g., random function, counting technique, etc.) to solve agreement problem. Agreement is a fundamental problem in distributed computing including a family of abstractions. In this thesis, we mainly focus on the design of consensus, set agreement, broadcast algorithms in anonymous and homonymous distributed systems. Firstly, the fault-tolerant broadcast abstraction is studied in anonymous systems with reliable or fair lossy communication channels separately. Two classes of anonymous failure detectors AΘ and AP∗ are proposed, and both of them together with a already proposed failure detector ψ are implemented and used to enrich the system model to implement broadcast abstraction. Then, in the study of the consensus abstraction, it is proved the AΩ′ failure detector class is strictly weaker than AΩ and AΩ′ is implementable. The first implementation of consensus in anonymous asynchronous distributed systems augmented with AΩ′ and where a majority of processes does not crash. Finally, a general consensus problem– k-set agreement is researched and the weakest failure detector L used to solve it, in asynchronous message passing systems where processes may crash and recover, with homonyms (i.e., processes may have equal identities), and without a complete initial knowledge of the membership.

Agreement Abstractions in Anonymous and Homonymous Distributed Systems Prone to Failures

The distributed computing models typically assume every process in the system has a distinct identifier (ID) or each process is programmed differently, which is named as eponymous system. In such kind of distributed systems, the unique ID is helpful to solve problems: it can be incorporated into messages to make them trackable (i.e., to or from which process they are sent) to facilitate the message transmission; several problems (leader election, consensus, etc.) can be solved without the information of network property in priori if processes have unique IDs; messages in the register of one process will not be overwritten by others process if this process announces; it is useful to break the symmetry. Hence, eponymous systems have influenced the distributed computing community significantly either in theory or in practice. However, every thing in the world has its own two sides. The unique ID also has disadvantages: it can leak information of the network(size); processes in the system have no privacy; assign unique ID is costly in bulk-production(e.g, sensors). Hence, homonymous system is appeared. If some processes share the same ID and programmed identically is called homonymous system. Furthermore, if all processes shared the same ID or have no ID is named as anonymous system. In homonymous or anonymous distributed systems, the symmetry problem (i.e., how to distinguish messages sent from which process) is the main obstacle in the design of algorithms. This thesis is aimed to propose different symmetry break methods (e.g., random function, counting technique, etc.) to solve agreement problem. Agreement is a fundamental problem in distributed computing including a family of abstractions. In this thesis, we mainly focus on the design of consensus, set agreement, broadcast algorithms in anonymous and homonymous distributed systems. Firstly, the fault-tolerant broadcast abstraction is studied in anonymous systems with reliable or fair lossy communication channels separately. Two classes of anonymous failure detectors AΘ and AP∗ are proposed, and both of them together with a already proposed failure detector ψ are implemented and used to enrich the system model to implement broadcast abstraction. Then, in the study of the consensus abstraction, it is proved the AΩ′ failure detector class is strictly weaker than AΩ and AΩ′ is implementable. The first implementation of consensus in anonymous asynchronous distributed systems augmented with AΩ′ and where a majority of processes does not crash. Finally, a general consensus problem– k-set agreement is researched and the weakest failure detector L used to solve it, in asynchronous message passing systems where processes may crash and recover, with homonyms (i.e., processes may have equal identities), and without a complete initial knowledge of the membership.

Análisis de la marcha: evaluación de un exoesqueleto aplicado a la marcha asistida

La marcha humana es el mecanismo de locomoción por el cual el cuerpo humano se traslada en línea recta gracias a una serie de movimientos coordinados de la pelvis y de las articulaciones del miembro inferior. Frecuentemente se encuentra influenciada por factores biomecánicos, anatómicos o patologías del sistema neuromusculoesquelético que modifican la forma de caminar de cada individuo. La lesión de médula espinal es una de las patologías que afectan el desarrollo normal de los patrones de la marcha por alteración de la movilidad, de la sensibilidad o del sistema nervioso autónomo. Aunque la lesión medular afecta otras funciones, además de la pérdida de función motora y sensorial, la recuperación de la capacidad de caminar es la mayor prioridad identificada por los pacientes durante la rehabilitación. Por ello, el desarrollo de dispositivos que faciliten la rehabilitación o compensación de la marcha es uno de los principales objetivos de diferentes grupos de investigación y empresas. En el contexto del proyecto Hybrid Technological Platform for Rehabilitation, Functional Compensation and Training of Gait in Spinal Cord Injury Patients se ha desarrollado un dispositivo que combina una órtesis activa (exoesqueleto) y un andador motorizado. Este sistema, como otros dispositivos, tiene el movimiento humano como estándar de referencia, no obstante no se evalúa de manera habitual, cómo es el patrón de la marcha reproducido y su similitud o diferencias con la marcha humana, o las modificaciones o adaptaciones en la interacción con el cuerpo del paciente. El presente estudio trata de examinar las características de la marcha normal en diversos grupos de población, y las diferencias con el patrón de marcha lenta. Finalmente, se pretende evaluar qué modificaciones y adaptaciones sufre el patrón de marcha lenta teórico al ser reproducido por el exoesqueleto. La presente investigación consiste en un estudio cuantitativo transversal desarrollado en dos etapas: estudio 1 y estudio 2. En el estudio 1 se analizó el patrón de la marcha a velocidad libremente seleccionada (normal) y el patrón de la marcha a velocidad lenta (0.25m/s) en 62 sujetos distribuidos en grupos considerando el sexo y los percentiles 25, 50 y 75 de estatura de la población española. Durante el estudio 2 se analizó el patrón de la marcha lenta reproducido por el dispositivo Hybrid a diferentes porcentajes de peso corporal (30%, 50% y 70%) en diez sujetos seleccionados aleatoriamente de la muestra del estudio 1. En ambos estudios se obtuvieron variables espacio-temporales y cinemáticas mediante un sistema de captura de movimiento con 6 cámaras distribuidas a lo largo de un pasillo de marcha. Se calcularon las medias, las desviaciones estándar y el 95% de intervalo de confianza, y el nivel alfa de significación se estableció en α=0.05 para todas las pruebas estadísticas. Las principales diferencias en el patrón normal de la marcha se encontraron en los parámetros cinemáticos de hombres y mujeres, aunque también se presentaron diferencias entre los grupos en función de la estatura. Las mujeres mostraron mayor flexión de cadera y rodilla, y mayor extensión de tobillo que los hombres durante el ciclo normal, aunque la basculación lateral de la pelvis, mayor en las mujeres, y el desplazamiento lateral del centro de gravedad, mayor en los hombres, fueron los parámetros identificados como principales discriminantes entre sexos. La disminución de la velocidad de la marcha mostró similares adaptaciones y modificaciones en hombres y en mujeres, presentándose un aumento de la fase de apoyo y una disminución de la fase de oscilación, un retraso de los máximos y mínimos de flexoextensión de cadera, rodilla y tobillo, y una disminución del rango articular en las tres articulaciones. Asimismo, la basculación lateral de la pelvis y el movimiento vertical del centro de gravedad disminuyeron, mientras que el movimiento lateral del centro de gravedad y el ancho de paso aumentaron. Durante la evaluación del patrón de la marcha reproducido por el exoesqueleto se observó que las tres articulaciones del miembro inferior disminuían el rango de movimiento por la falta de fuerza de los motores para contrarrestar el peso corporal, incluso con un 70% de descarga de peso. Además, la transferencia de peso se encontró limitada por la falta de movimiento de la pelvis en el plano frontal y se sustituyó por un aumento de la inclinación del tronco y, por tanto, del movimiento lateral del centro de gravedad. Este hecho, junto al aumento del desplazamiento vertical del centro de gravedad, hizo del patrón de la marcha reproducido por el exoesqueleto un movimiento poco eficiente. En conclusión, se establecen patrones de marcha normal diferenciados por sexos, siendo la basculación lateral de la pelvis y el movimiento lateral del centro de gravedad los parámetros discriminantes más característicos entre sexos. Comparando la marcha a velocidad libremente seleccionada y la velocidad lenta, se concluye que ambos sexos utilizan estrategias similares para adaptar el patrón de la marcha a una velocidad lenta y se mantienen las características diferenciadoras entre hombres y mujeres. En relación a la evaluación del dispositivo Hybrid, se deduce que la falta de movimiento lateral de la pelvis condiciona la transferencia de peso y el aumento del rango de movimiento del centro de gravedad y, en consecuencia, tiene como resultado un patrón de la marcha poco eficiente. Este patrón no resultaría indicado para los procesos de rehabilitación o recuperación de la marcha, aunque podría considerarse adecuado para la compensación funcional de la bipedestación y la locomoción. ABSTRACT The human walking is a means of moving body forward using a repetitious and coordinated sequence of pelvis and lower limb motions. It is frequently influenced by biomechanical and anatomical factors or by musculoskeletal pathologies which modify the way of walking. The spinal injury is one of those pathologies which affect the normal pattern of walking, due to the alteration of the mobility, the sensory or the autonomic nervous system. Although the spinal injury affects many other body functions, apart from the motor and sensory ones, the main priority for patients is to recover the ability of walking. Consequently, the main objective of many research groups and private companies is the development of rehabilitation and compensation devices for walking. In this context, the Hybrid Technological Platform for Rehabilitation, Functional Compensation and Training of Gait in Spinal Cord Injury Patients project has developed a device which integrates an exoskeleton and a motorized smart walker. This system, as other similar devices, has the human movement as standard reference. Nevertheless, these devices are not usually evaluated on the way they reproduce the normal human pattern or on the modifications and in the interactions with the patient’s body. The aim of the present study is to examine the normal walking characteristics, to analyze the differences between self-selected and low speed walking patterns, and to evaluate the modifications and adaptations of walking pattern when it is reproduced by the exoskeleton. The present research is a quantitative cross-sectional study carried out in two phases: study 1 and study 2. During the study 1, the self-selected and the low speed (0.25m/s) walking patterns were analyzed in sixty-two people distributed in groups, according to sex and 25th, 50th and 75th percentiles of height for Spanish population. The study 2 analyzed the low speed walking pattern reproduced by the Hybrid system in three conditions: 30%, 50% and 70% of body weight support. To do this, ten subjects were randomly selected and analyzed from the people of study 1. An optoelectronic system with six cameras was used to obtain spatial, temporal and kinematic parameters in both studies. Means, standard deviations and 95% confidence intervals of the study were calculated. The alpha level of significance was set at α=0.05 for all statistical tests. The main differences in normal gait pattern were found in kinematic parameters between men and women. The hip and the knee were more flexed and the ankle plantar flexion was higher in women than in men during normal gait cycle. Although the greater pelvic obliquity of women and the higher lateral movement of center of gravity of men were the most relevant discriminators between male and female gait patterns. Comparing self-selected and low speed walking patterns, both sexes showed similar adaptations and modifications. At low speed walking, men and women increased the stance phase ratio and decreased the swing phase ratio. The maximum and minimum peak flexion of hip, knee and ankle appeared after and the range of motion of them decreased during low speed walking. Furthermore, the pelvic obliquity and the vertical movement of the center of gravity decreased, whereas the lateral movement of center of gravity and step width increased. Evaluating the gait pattern reproduced by the exoskeleton, a decrease of lower limb range of motion was observed. This was probably due to the lack of strength of the engines, which were not able to control the body weight, even with the 70% supported. Moreover, the weight transfer from one limb to the contralateral side was restricted due to the lack of pelvis obliquity. This movement deficiency was replaced by the lateral torso sway and, consequently, the increase of lateral movement of the center of gravity. This fact, as well as the increase of the vertical displacement of the center of gravity, made inefficient the gait pattern reproduced by the exoskeleton. In conclusion, different gait patterns of both sexes have been determined, being pelvis obliquity and lateral movement of center of gravity the most relevant discriminators between male and female gait patterns. Comparing self-selected and low speed walking patterns, it was concluded that both sexes use similar strategies for adapting the gait pattern to a low speed, and therefore, the differentiating characteristics of normal gait are maintained. Regarding the Hybrid system evaluation, it was determined that the gait pattern reproduced by the exoskeleton is inefficient. This was due to the lack of pelvis obliquity and the increase of the center of gravity displacement. Consequently, whereas the walking pattern reproduced by the exoskeleton would not be appropriated for the rehabilitation process, it could be considered suitable for functional compensation of walking and standing.

Agrobacterium VirD2 protein interacts with plant host cyclophilins

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5′ end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2–cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for Gαi subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC–a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.

The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3–10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3–10 reversion rate, whereas the hom3–10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor

Several G-protein coupled receptors, such as the β1-adrenergic receptor (β1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein–protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the β1-AR either as a glutathione S-transferase fusion protein in biochemical “pull-down” assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the β1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the β1-AR but not to that of the β2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of β1-ARs in HEK293 cells while having no effect on β2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in β1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Protein–protein interactions among the Aux/IAA proteins

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic βαα DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the βαα containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis λ-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1–1 mutation. We now describe a third suppressor of sec1–1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of Gαi subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein Gαi3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP—a soluble and a membrane-anchored pool—were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the Gαi subfamily, Gαi1, Gαi2, Gαi3, Gαz, and Gαo, but not with members of other Gα subfamilies, Gαs, Gαq, and Gα12/13. The C terminus of Gαi3 is important for binding because a 10-aa C-terminal truncation and a point mutant of Gαi3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of Gαi3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP’s possible involvement in membrane trafficking.