Absence of annulus in human asthenozoospermia: case report.

The annulus is a septin-based ring structure located at the junction of the midpiece (MP) and the principal piece (PP) of spermatozoa flagellum. In the mouse, deletion of Septin 4, a structural component of the sperm annulus, prevents annulus formation and leads to MP-PP disjunction, flagellar bending, asthenozoospermia and male sterility. Testis anion transporter 1 (Tat1) is a germ cell-specific member of the SLC26 anion transporter family and is co-expressed with Septin 4 at the sperm annulus. Interestingly, Tat1 null sperm bear an atrophic annulus, causing a phenotype similar to that of Sept4 null sperm. We searched for Tat1 misexpression and/or mislocalization in spermatozoa from asthenozoospermic subjects (n = 75) and controls by performing an immunofluorescence detection assay on sperm smear preparations. We found one patient showing moderate asthenozoospermia, with 97% of sperm lacking Tat1, Septin 4 and Septin 7 proteins at the annulus. We confirmed the absence of the annulus structure by transmission electron microscopy and observed that spermatozoa from the patient displayed MP-PP disjunction and abnormal mitochondrial organization. We show that the structural defects in sperm are not caused by abnormal transcription or point mutations of the TAT1 and SEPT4 genes; however, although both proteins are expressed, they are not properly localized at sperm annulus. The case we studied, so far unreported in human, confirms the involvement of Tat1 and Septin proteins in the constitution of the annulus, but also raises questions about the function of this structure in human sperm motility.

[Metaphyseal and diaphyseal modular femoral stems implanted without cement]. / Modularité métaphyso-diaphysaire et tiges fémorales sans ciment.

PURPOSE OF THE STUDY: We assessed outcome after total hip arthroplasty (THA) using a dual metaphyseal-diaphyseal modular femoral stem with hydroxyapatite coating on the metaphyseal portion only. Implanted without cement, this stem was adaptable to all the anatomic morphotypes defined by the Noble canal flare index. MATERIAL AND METHODS: THA was performed in 116 patients (124 hips), mainly for degenerative joint disease (80% for dysplasia). Mean age was 61.2 years. Follow-up was 6.9 years (72-108 months). RESULTS: There were no preoperative complications excepting 3 cases of neck fissuration without clinical consequence. There was no trochanteric fracture. We had two early and one late dislocations. The Postel Merle d'Aubigné score improved from 8.09 to 17.27. Clinical outcome was not influenced by patient age, weight or morphotype. Radiologically, signs of bone ingrowth were found in more than 80% of the cases. Lucent lines were seen in only 3 cases. There was a single case of migration. No revision was needed among the cases with ossifications (23%, 22% Brooker I) and no femur revisions were required. There was no mechanical problem involving the metaphyseal-epiphysial junction. DISCUSSION: The dual metaphyseal-diaphyseal modular stem was found to be a safe and effective implant adaptable to all anatomic variations of the femur and providing good primary stability. In our series, 58% of the femurs were "standard" but for one-third of the femurs, the modular stem enabled implantation without cement, particularly in young adults with a dysplasic or funnel-shaped femur. CONCLUSION: The dual metaphyseal-diaphyseal modular stem was found to be most useful for optimizing total hip arthroplasty without cement.

[Single-compartment knee arthroplasty prosthesis and idiopathic necrosis of the medial tibia surface]. / Prothèse unicompartimentaire et nécrose idiopathique du plateau tibial interne.

PURPOSE OF THE STUDY: Idiopathic necrosis of the medial articular surface of the tibia is exceptional. Diagnosis is quite difficult and often made late. Among the different treatments proposed, we preferred single-compartment arthroplasty. MATERIAL AND METHODS: We report 8 cases in women with a mean age of 71.1 years. Diagnosis was suspected due to drug-resistant knee pain, particularly frequent at night, initially with radiographically normal knees. The first radiographic signs, seen 3 months after the onset of pain, were pathognomonic for osteonecrosis evidencing subchondral defects of the tibial surface with a dense peripheral rim and apparently "sequestered" in a notch. Bone scintigraphy evidenced intense uptake in the medial compartment. MRI confirmed the diagnosis evidencing a band of low intensity signals completely surrounding a sequestered zone reaching the cortical. This band was stable and irreversible. In 5 cases CT scan and in 3 cases tomography identified the width and height of the necrotic area that was limited to the medial compartment in all cases. All patients were treated with a single compartment implant. The diagnosis of necrosis was confirmed at pathology. RESULTS: At 4,6 years of mean follow up all patients had an excellent outcome, "forgetting" their knee. No lucent lines developed along the femoral or tibial implants. DISCUSSION: Necrosis of the medial articular surface of the tibia is exceptional and often diagnosed late by bone scintigraphy or MRI. Surgical treatment is usually based on tibial osteotomy for valgisation or a single or three-compartment prosthesis. In our 8 cases, the necrosis was limited to the medial compartment, warranting our therapeutic option.

Tat1, a novel sulfate transporter specifically expressed in human male germ cells and potentially linked to rhogtpase signaling.

RhoGTPases (Rho, Rac, and Cdc42) are known to regulate multiple functions, including cell motility, adhesion, and proliferation; however, the signaling pathways underlying these pleiotropic effects are far from fully understood. We have recently described a new RhoGAP (GTPase activating protein for RhoGTPases) gene, MgcRacGAP, primarily expressed in male germ cells, at the spermatocyte stage. We report here the isolation, through two-hybrid cloning, of a new partner of MgcRacGAP, very specifically expressed in the male germ line and showing structural features of anion transporters. This large protein (970 amino acids and a predicted size of 109 kDa), we provisionally designated Tat1 (for testis anion transporter 1), is closely related to a sulfate permease family comprising three proteins in human (DRA, Pendrin, and DTD); it is predicted to be an integral membrane protein with 14 transmembrane helices and intracytoplasmic NH(2) and COOH termini. In situ hybridization studies demonstrate that Tat1 and MgcRacGAP genes are coexpressed in male germ cells at the spermatocyte stage. On testis sections, Tat1 protein can be immunodetected in spermatocytes and spermatids associated with plasma membrane. Two-hybrid and in vitro binding assays demonstrate that MgcRacGAP stably interacts through its NH(2)-terminal domain with the Tat1 COOH-terminal region. Expression of Tat1 protein in COS7 cells generates a 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene and chloride-sensitive sulfate transport. Therefore we conclude that Tat1 is a novel sulfate transporter specifically expressed in spermatocytes and spermatids and interacts with MgcRacGAP in these cells. These observations raise the possibility of a new regulatory pathway linking sulfate transport to Rho signaling in male germ cells.

[Osteolysis after total knee prosthesis]. / Ostéolyse après prothèses totales du genou.

PURPOSE OF THE STUDY: The aim of our work was to study X-rays showing osteolysis after 5 years and more in 122 prosthesis and to try and assess such complication, often described in the United States but seldom in Europ. MATERIAL: We are dealing here with 122 retaining posterior cruciate ligament, mostly cementless prothesis implanted between 1985 and 1992 84 chromium-cobalt prosthesis (PCA and Themis) implanted in 34 males and 88 females with an average age of 67 (45-81), 87,7 p. 100 had femoral cementless components and 70 p. 100 tibial cementless components. METHODS: All patients were examined and had X-rays at an average of 6,9 years. Specially considered were X-rays showing a possible osteolysis. We looked for possible complication (external laxity, anterior femoral dislocation and polyethylene wear), assessment of the mechanical axis and for clinical results (Hungerford score) RESULTS: Revisions: 19 arthroplasties were revised for PE wear tibial loosening metallosis or patella problems The postoperative score according to Hungerford was 84,5 p. 100 for PCA prosthese and 87 p.100 for the Themis. On the X-rays were only few osteolysis to be found: 9 cases (7,3 p. 100). For the PCA series: 3 femoral osteolysis, 1 tibial at 12 years, and one patellar osteolysis. For the Themis series: no femoral osteolysis, 3 tibial and one patellar osteolysis. Osteolysis are apparent on X-rays in profile for the femur and the patella, and in both profile and frontal X-rays for the tibia. Clinicaly 4 osteolysis really asymptomatic were not re-operated. 5 were revised: one 11 years later for femoral and tibial loosening, two for a patellar loosening, the other two patients had to be reoperated on for metallism (titanium's femoral component) and for those two instances osteolysis were discovered during the complication. DISCUSSION: Osteolysis after TKA appears unusual in our experience without bearing on frequency finded by american authors with a lesser follow-up (Engh 11,1 p. 100 after 4,5y, Peters 16 p. 100 after 2,9 y, Robinson 9,18 p. 100 after 4,6 y). American litteratur analysis shows that the important number of osteolysis is due to: - either to dual-metal using (Co-Cr component with Titanium screws for exemple), - or bad quality of polyethylene (compressed), - or a bad design of former prosthesis. CONCLUSION: Interface illness, linked to the production of wear debris, osteolysis after total knee arthroplasty is rarer than after a hip one, probably because size of debris is different, larger in knee than in hip. It is likely that the improvement of PE quality, design of prosthesis, as well as a better knowledge of osteolysis mechanism will allow to delay this complication wich is in a long term ineluctable.

Structure and expression of murine mgcRacGAP: its developmental regulation suggests a role for the Rac/MgcRacGAP signalling pathway in neurogenesis.

Rho-family GTPases regulate a wide range of biological functions including cell migration, cell adhesion and cell growth. Recently, results from studies in vivo in Drosophila, mouse and humans have demonstrated the involvement of these GTPases in mechanisms controlling neuronal differentiation and the development of the central nervous system (CNS). However, the signalling pathways underlying these functions and the proteins directly regulating RhoGTPases in developing neurons are poorly defined. Here we report the structure and expression pattern of the murine orthologue of mgcRacGAP, a human gene encoding a RacGTPase partner expressed in male germ cells [Touré, Dorseuil, Morin, Timmons, Jegou, Reibel and Gacon (1998) J. Biol. Chem. 273, 6019-6023]. In contrast with that from humans, murine mgcRacGAP encodes two distinct transcripts. Both are developmentally regulated. A 2.2 kb transcript is strongly expressed in mature testis and is up-regulated with spermatogenesis. A 3 kb RNA is predominant in the embryo and is expressed primarily in the CNS during the neurogenic phase, decreasing after birth. In situ hybridization analysis in embryonic-day 14.5 mouse embryos demonstrates a preferential expression of mgcRacGAP in the proliferative ventricular zone of the cortex. In addition to the expression of mgcRacGAP in male germ cells already reported in humans and suggesting an involvement in spermatogenesis, we characterize an embryonic transcript whose expression is closely correlated with neurogenesis. This result addresses the question of the role of Rac/MgcRacGAP pathway in neuronal proliferation.

MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells.

In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases. A specific mRNA of 3.2 kilobases was detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we call MgcRacGAP, was detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific to recombinant protein. In vitro, the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs. The closest homolog of MgcRacGAP is RotundRacGAP, a product of the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility.

Enhanced apoptosis in the thymus of transgenic mice expressing constitutively activated forms of human Rac2GTPase.

Rac proteins constitute a subgroup of the Rho family of small GTPases and include Rac1, which is expressed ubiquitously, and Rac2, a highly homologous protein only expressed in myelo-monocytic and lymphoid cell lineages. In fibroblasts, Rac1 plays a crucial role in control of actin cytoskeleton organisation, cell growth and Ras-induced transformation. In phagocytes, Rac1 and Rac2 regulate a specific enzymatic complex, NADPH oxidase. These multiple functions have been ascribed to Rac proteins only on the basis of cell culture and in vitro biochemical studies. To examine the role of Rac2 in vivo in a T cell lineage, we have expressed either wild-type or constitutively-activated forms of human Rac2 (Rac2V12 and Rac2L61) in transgenic mice under control of the thymus specific lck proximal promoter. We report here a striking atrophy of the thymus in mice expressing even low levels of either of the activated mutants of Rac2, while expression of Rac2wt has no effect. This phenotype is correlated with a marked decrease in the number of double positive (CD4+ CD8+) and single positive (CD4+ CD8- and CD8+ CD4-) thymocytes. Cellular and molecular analyses demonstrate that this defect is due to an increase in apoptosis among thymocytes. As Rac2 is normally expressed in thymocytes and activated T cells, we propose that Rac2 dependent pathways could play an important role in control of growth and death of T cells.

[Signal transduction by Rac small G proteins in phagocytes]. / Signalisation par les petites protéines G de la famille Rac dans les phagocytes.

Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro, however, Rac2 is largely predominant in human phagocytes. NADPH oxidase is a plasma membrane enzyme of phagocytes, generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme, minimally requires assembly at the membrane with flavocytochrome b258 of cytosolic components p47phox, p67phox and Rac proteins. Using the yeast two hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants which are known to be inactive in NADPH oxidase lose their interaction with p67phox. Finally, we observe that p67phox interacts six fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac, and particularly Rac2, can regulate superoxide production by NADPH oxidase of phagocytic cells through direct interaction with p67phox subunit. Recently published data suggest that Rac proteins could transduce mitogenic signals in non-phagocytic cells through superoxide production by a phagocytic-related NADPH oxidase enzymatic system which remains to be determined. NADPH oxidase regulation by Rac proteins in phagocytes could then be used as a model to understand the molecular mechanisms underlying Rac functions in various cell types.

[Two stages reimplantation for infection after knee arthroplasty. Apropos of a series of 29 cases]. / Réimplantation en deux temps pour infection après arthroplastie du genou. A propos d'une série de 29 cas.

PURPOSE OF THE STUDY: The purpose of this work was to precise diagnosis and treatment of infected total knee arthroplasty with two stage reimplantation. MATERIAL: 29 infected total knee arthroplasties were operated between 1984 and 1994 and included in this study (mean F.U. 3.5 Y). There were 20 females and 9 males, mean age 70 (46-83). The original arthroplasty was done for OA in 28 patients, RA in one. The arthroplasties were: UHK 2, Bicompartmental 2, Tricompartmental 19. 20 TKA were cementless. 14 patients showed one or several risk factors. Infection was diagnosed in 1 of 2 ways: preoperative aspiration or culture of surgical specimen. There were 12 staphylococcus epidermidis, 8 staphylococcus aureus, streptococcus (n = 2) acinetobacter (n = 2), peptococcus (n = 1) pseudomonas (n = 1), gemella morbidellum (n = 1). 6 were non identified. METHOD: The protocol for two stage reimplantation began with components and cement removal. A synovectomy was performed. The knee cavity was filled with antibiotic cement spacer and the wound was closed. The leg was placed in a splint. All patients underwend a continued antibiotic therapy, specific in 20 cases with isolated organisms. A total knee arthroplasty was performed, using a total posterior cruciate substituting prosthesis, 6 to 8 weeks after components removal (2-24). All patients received parenteral antibiotics after reimplantation for not less than 2 months (2-6). RESULTS: Infection was eradicated in 24 cases, 22 in one time, 2 bad second debridement. At last follow-up the average Hungerford score was 75.6/100, the average Knee society knee score was 80 and the average functional score was 70. Mean range of flexion was 95 degrees. 6 patients had recurrent infection and poor result. They underwent arthrodesis. 5 of the 6 patients had solid mature fusion at last follow-up. DISCUSSION: The results of two stage reimplantation for infected total knee replacement showed that this is the method of choice for infection treatment and acceptable function restoration. As other authors, we get a good success rate (82 per cent). Functional result was better with identified microorganisms, but we did not find any correlation with organisms type or infection length. Punction and bone scanning are of great help for diagnosis in difficult chronical cases. Organism identification is fundamental for infection duration. Staphylococcus epidermidis was the most frequent identified organism. New procedures using articulated cement spacer may improve functional results.

The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1.

NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b558 of cytosolic components p47phox, p67phox, and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47phox, p67phox, and Rac proteins. Rac proteins interact with p67phox in a GTP-dependent manner, but do not interact with p47phox. Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67phox; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67phox. Finally, we observe that p67phox interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47phox and p67phox. These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67phox and that Rac2 is the main p67phox-interacting GTPase in human cells.

Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity.

Ra1A and Ra1B are GTPases of unknown function and are activated by proteins, Ra1GDS, that interact with the active form of another GTPase, Ras. To elucidate Ral function, we have searched for proteins interacting with an activated form of Ra1A using the two-hybrid method and a Jurkat cell library. We have identified a partial cDNA encoding a protein, RLIP1, which binds to activated Ra1A and this binding requires an intact effector domain of Ra1A. Biochemical data with purified Ra1A confirm the genetic results. This protein also bears a region of homology with GTPase-activating protein (GAP) domains that are involved in the regulation of GTPases of the Rho family and, indeed, RLIP1 displays a GAP activity acting upon Rac1 and CDC42, but not RhoA. This GAP region is not required for RLIP1 binding to Ra1. The whole cDNA was cloned, and it encodes a 76-kDa polypeptide, RLIP76, which also binds RalA. The Rho pathway is involved in membrane and cytoskeleton modifications after mitogenic stimulation and acts in parallel to and synergistically with the Ras pathway. We propose that these pathways are linked through a cascade composed of Ras --> Ra1GDS --> Ra1 --> RLIP76 --> CDC42/Rac1/Rho, allowing modulation of the Rho pathway by the Ras pathway.