IntAct is an open-source, open data molecular interaction database and toolkit. Data is abstracted from the literature or from direct data depositions by expert curators following a deep annotation ...model providing a high level of detail. As of September 2009, IntAct contains over 200.000 curated binary interaction evidences. In response to the growing data volume and user requests, IntAct now provides a two-tiered view of the interaction data. The search interface allows the user to iteratively develop complex queries, exploiting the detailed annotation with hierarchical controlled vocabularies. Results are provided at any stage in a simplified, tabular view. Specialized views then allows 'zooming in' on the full annotation of interactions, interactors and their properties. IntAct source code and data are freely available at http://www.ebi.ac.uk/intact.
An abstract of the study by Williams et al performing a high-throughput combinatorial siRNA screen of the human kinome across a panel of 33 colorectal cancer cell lines to identify synthetic lethal ...combinations and potential targets for therapy is presented. In this study, the screen utilised inhibitors of EGFR, MEK and PI3K in combination with the siRNA library to identify synergistic gene interaction events. Genomic and proteomic data were utilised in order to identify biomarker signatures that could be used to stratify patient populations that are predicted to be sensitive to these novel combinations.
Summary Background Patellofemoral instability following total knee arthroplasty is a very common complication which may result from a defective rotational positioning of the femoral component. ...However, rotational landmarks for optimal orientation are not unequivocal. Moreover, no proven correlation has yet been established between preexisting rotational malposition and patellofemoral instability occurrence. Hypothesis Any preexisting distal femoral rotational misalignment is associated with a preop patellofemoral instability in arthritic knees prior to undergoing arthroplasty. A prospective diagnostic study was conducted to test this hypothesis on the basis of morphometric data. Material and methods One hundred and eighteen patients were prospectively enrolled in this study. Patellar lateralization was measured on 30̊ flexion patellofemoral views. Three positionings were arbitrarily defined (less than 3 mm of lateralization, between 3 and 5 mm, over 5 mm). Three angles were preoperatively measured using CT scans: (1) the posterior condylar angle between posterior bicondylar axis and transepiphyseal axis, (2) the anterior trochlear angle between transepicondylar axis and trochlear opening plane, (3) the sum of anterior trochlear and posterior condylar angles finally formed the global trochlear opening angle. Results The patella was centered in 86 cases and lateralized in 32 cases (less than 5 mm in 25 cases and over 5 mm in seven cases). Independently from the degree of patellar lateralization, the global trochlear opening angle was constant ( p = 0,41). The value of the posterior condylar angle was statistically inferior when patella was centered ( p = 0,01; r = 0,44). The value of the anterior trochlear angle varied opposite to the posterior condylar angle. Femoral anteversion, position of the anterior tibial tuberosity and tibiofemoral index could not be correlated with patellar positioning. No relationship could be established between patellar lateralization and overall torsional deformities of the lower extremity. Conclusion The centering of the patella in arthritic knees depends on distal femoral osseous factors which determines the posterior condylar angle and anterior trochlear angle on either side of the transepicondylar axis. Since the trochlear opening angle is constant, the obliquity of the transepicondylar axis appears crucial in patellar lateralization. A better understanding of the influence of distal femoral morphology on patellar positioning will ensure improved positioning of femoral components in total knee arthroplasties or in isolated femoropatellar joint replacements. Level of Evidence III: Prospective diagnostic study.
The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent ...exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that α-soluble NSF-attachment protein (α-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of α-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-α-SNAP antibodies. Third, bacterially expressed α-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for α-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for α-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.
The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and
the cell membrane. In spite of the great amount of information available ...related to the acrosome reaction in several species,
there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab familyâwell-established
participants in exocytosis in other cell typesâin the acrosome reaction. Western blot and immunofluorescence analysis indicate
that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying
the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established
a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated
way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide
corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis.
The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)âa
protein that releases Rab proteins from membraneâinhibited a GTPγS-stimulated acrosome reaction. Our results indicate that
1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present
in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human
spermatozoa.
The acrosome reaction of spermatozoa is a complex, calcium-dependent, regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of ...the acrosomal contents and the loss of the membranes surrounding the acrosome. However, very little is known about the molecules that mediate and regulate this unique fusion process. Here, we show that N-ethylmaleimide-sensitive factor (NSF), a protein essential for most fusion events, is present in the acrosome of several mammalian spermatozoa. Moreover, we demonstrate that calcium-dependent exocytosis of permeabilized sperm requires active NSF. Previously, we have shown that the addition of the active (GTP-bound) form of the small GTPase Rab3A triggers exocytosis in permeabilized spermatozoa. In the present report we show that Rab3A is necessary for calcium-dependent exocytosis. The activation of Rab3A protects NSF from N-ethylmaleimide inhibition and precludes the exchange of the endogenous protein with recombinant dominant negative mutants of NSF. Furthermore, Rab3A activation of acrosomal exocytosis requires active NSF. Our results suggest that, upon calcium stimulation, Rab3A switches to its active GTP-bound form, triggering the formation of a protein complex in which NSF is protected. This process is suggested to be an essential part of the molecular mechanism of membrane fusion leading to the release of the acrosomal contents.
MARCKS (myristoylated alanine-rich C-kinase substrate) is a major substrate for protein kinase C (PKC), a kinase that has multiple functions during oocyte maturation and egg activation, for example, ...spindle function and cytoskeleton reorganization. We examined temporal and spatial changes in p-MARCKS localization during maturation of mouse oocytes and found that p-MARCKS is a novel centrosome component based its co-localization with pericentrin and γ-tubulin within microtubule organizing centers (MTOCs). Like pericentrin, p-MARCKS staining at the MI spindle poles was asymmetric. Based on this asymmetry, we found that one end of the spindle was preferentially extruded with the first polar body. At MII, however, the spindle poles had symmetrical p-MARCKS staining. p-MARCKS also was enriched in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain. Because phosphorylation of MARCKS modulates its actin crosslinking function, this localization suggests p-MARCKS functions as part of the contractile apparatus during polar body emission. Our finding that an activator of conventional and novel PKC isoforms did not increase the amount of p-MARCKS suggested that an atypical isoform was responsible for MARCKS phosphorylation. Consistent with this idea, immunostaining revealed that the staining patterns of p-MARCKS and the active form of the atypical PKC ζ/λ isoform(s) were very similar. These results show that p-MARCKS is a novel centrosome component and also defines a previously unrecognized subdomain of the actin cap overlying the spindle.
B cell development depends on the coordinated expression and cooperation of several transcription factors. Here we show that the transcription factor ETS-related gene (ERG) is crucial for normal B ...cell development and that its deletion results in a substantial loss of bone marrow B cell progenitors and peripheral B cells, as well as a skewing of splenic B cell populations. We find that ERG-deficient B lineage cells exhibit an early developmental block at the pre-B cell stage and proliferate less. The cells fail to express the immunoglobulin heavy chain due to inefficient V-to-DJ recombination, and cells that undergo recombination display a strong bias against incorporation of distal V gene segments. Furthermore, antisense transcription at PAX5-activated intergenic repeat (PAIR) elements, located in the distal region of the Igh locus, depends on ERG. These findings show that ERG serves as a critical regulator of B cell development by ensuring efficient and balanced V-to-DJ recombination.
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•Loss of the transcription factor ERG leads to an early block in B cell development•ERG is required for Igh V-to-DJ recombination involving distal V segments•ERG is an important transcriptional regulator of B cell development
Søndergaard et al. demonstrate that ERG is a critical transcriptional regulator essential for B cell development. Loss of ERG leads to a marked reduction in V-to-DJ recombination at the Igh locus, precluding B cell progenitors from expressing the pre-B cell receptor, which is required for entering the final stages of B cell development.
The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in ...permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.
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