The IntAct molecular interaction database in 2010.

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.

Distal femur rotational alignment and patellar subluxation: a CT scan in vivo assessment.

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 30 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.

[Distal femoral torsion: comparison of CT scan and intra operative navigation measurements during total knee arthroplasty. A report of 70 cases]. / Torsion distale fémorale du genou : comparaison de la mesure par tomodensitométrie et par navigation peropératoire au cours des arthroplasties totales de genou. A propos de 70 cas.

INTRODUCTION: Proper positioning of the prosthesis components in total knee arthroplasty is an important factor for satisfactory outcomes such as mobility, pain and wear. If an independent cutting technique is performed, rotational alignment of the femoral component should adapt patient's specific anatomy. The distal epiphyseal femoral torsion (DEFT) is adjusted in order to align the prosthetic posterior condylar axis along the bone transepicondylar axis. The DEFT presents a high rate of interindividual variations. Computed tomography scanning produces reliable and reproducible measurement of the epiphyseal torsion, but this requires additional procedure. We therefore used intraoperative computed navigation for DEFT measurement, thus accurately adapting each patient's epiphyseal torsion during the procedure, without resorting to the preoperative CT scan. MATERIAL AND METHODS: This prospective study included 70 patients with arthritic knees who underwent TKA. Mean patient age was 74 years old. There were 52 women, 35 right knees, 33 genu varum, 19 genu valgum, 18 normal knees. DEFT was determined by preoperative CT scan, using the Yoshioka angle referencing. Distal epiphyseal femoral torsion measurement was carried out using the Navitrack system (Orthosoft). The DEFT was the navigated measured angle between the transepicondylar axis and the posterior condylar axis manually located and digitized with an optically tracked stylus. The navigation system was therefore used as a simple digital measurement device to evaluate the distal epiphyseal femoral torsion. We had established the reference transepicondylar axis as the line connecting the prominence of the medial and lateral epicondyles. During the procedure, we also carried out computer-assisted measurement of HKA axis in full extension and at 90 degrees of knee flexion. Correlation between the navigated HKA in full extension and HKA measured on the preoperative pangonogram (R(2)=0.621) demonstrated a high reliability of the navigation system in the frontal plane. RESULTS: There was no correlation between the mean epiphyseal torsion determined with computer navigation and the epiphyseal torsion measured on the CT scan (R(2)=0.09). Significant interindividual variations were reported. Navigated HKA at 90 degrees of knee flexion was not correlated with navigated HKA in full extension (R(2)=0.398) nor with epiphyseal torsion measured on the CT scan (R(2)=0.063). Results demonstrated a major interindividual variation. DISCUSSION: Our results report a large variability in distal epiphyseal femoral torsion measured with CT scan. Moreover, computed navigation does not provide a reliable and reproducible evaluation of the epiphyseal torsion. Due to inaccurate identification of femoral epicondyles, the related navigated measurement is not considered to be reliable and reproducible. Navigated HKA at 90 degrees of knee flexion is not a fair indirect reflection of epiphyseal torsion. Computer-assisted navigation fails to provide direct or indirect, reliable and reproducible intraoperative measurement of distal epiphyseal femoral torsion. Preoperative CT scan is the only reliable method to produce accurate measurement of distal epiphyseal femoral torsion.

[Rotational alignment of femoral component with computed-assisted surgery (CAS) during total knee arthroplasty]. / Rotation fémorale par navigation sur la base d'un scanner préopératoire dans l'arthroplastie totale de genou : à propos de 70 cas.

INTRODUCTION: Accurate implantation of the prosthesis components is a prognostic factor for long-term total knee arthroplasty survival as it reduces wear and loosening failure. Computer-assisted navigation systems have proved to produce accurate bone cuts orthogonal to the mechanical axis. Proper rotational alignment of the femoral component is one of the requirements for optimal positioning of the femoral prosthesis. The posterior bicondylar axis of the femoral prosthesis should therefore be parallel to the transepicondylar axis. The purpose of the present study was to determine whether computer-assisted navigation provides an accurate rotational alignment of the femoral implant, when preoperatively defined with CT scan. MATERIAL AND METHODS: This prospective study, carried out between December 2003 and June 2005, included 70 patients of average age 74 years old (range 57-85) who underwent primary total knee arthroplasty, with a SAL prosthesis (Zimmer). Preoperative investigations with computed tomography scanning produced accurate measurements of distal epiphyseal femoral torsion (DEFT). The posterior bicondylar axis was found to be a reliable landmark for the rotational orientation of the femoral cutting-guide during bone-cuts. The rotational orientation of the cutting-guide was based on the preoperative CT data. A three-month follow-up CT scan was carried out to evaluate final rotational position of the femoral component. RESULTS: The mean DEFT evaluated on the preoperative CT scan was 6.9+/-2.9 degrees . The mean rotational orientation of the cutting guide was 4.8+/-2 degrees . The mean postoperative measurement of DEFT was 1.56+/-2.7 degrees . The mean adjustment of DEFT was 5.34 degrees . Adopting a +/- 2 degrees cutoff, 77 % of patients achieved acceptable alignment within +/- 2 degrees compared with our objectives. These findings were compared to a previous series of 34 cases using an arbitrary 3 degrees standardized rotation of the femoral component and following an identical radiological protocol. Among the knees, 44% reported alignment within +/- 2 degrees . DISCUSSION: When femoral and tibial bone cuts are performed independently, conventional instrumentation techniques seem insufficient to adapt patient's specific anatomy and prove inadequate to provide precise rotational alignment of the femoral component. Computed tomography scan is a reliable mean to produce precise preoperative measurements for proper DEFT. Moreover, it allows accurate postoperative control of the implant positioning. Other studies have documented a higher degree of precision in the rotational alignment of the femoral component with computed navigation systems in comparison to conventional instrumentation. However, in such studies, rotational alignment was always determined by computer navigation, and based on a controversial intraoperative identification (epicondyles and Whiteside's line referencing). We believe that preoperative CT scanning is a more favourable method. Actually, 77% of the cases reported satisfactory rotational alignment of the femoral component using this technique.

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.

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 alpha-soluble NSF-attachment protein (alpha-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 alpha-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-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-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 alpha-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.

Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction.

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.

Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.

Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor.

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.

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa.

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 family-well-established participants in exocytosis in other cell types-in 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)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-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.