Hsp90{beta} and p130(cas): novel regulatory factors of MMP-13 expression in human osteoarthritic chondrocytes.

BACKGROUND: Human osteoarthritic (OA) chondrocytes were previously classified into L (low)- and H (high)-OA according to matrix metalloproteinase-13 (MMP-13) basal levels and interleukin 1beta (IL1beta) inducibility. In H-OA chondrocytes, the regulatory proteins p130(cas) and nuclear matrix protein 4 (NMP4) acting on the MMP-13 promoter were identified. OBJECTIVE: To identify regulators of MMP-13 expression/production in human L-OA chondrocytes, to determine their effect on the expression of other MMPs and the effect of IL1beta on these molecules. METHODS: The identification of the L-OA chondrocyte proteins interacting specifically with the AGRE site of the MMP-13 promoter was performed by mass spectrometry. Heat shock protein 90beta (Hsp90beta), p130(cas) and NMP4 small interfering RNAs (siRNAs) were transfected into L-OA chondrocytes and incubated with or without IL1beta. Gene expression was determined by real-time PCR, MMP-1 and MMP-13 production by ELISA, and signalling pathway activation by western blotting and ELISA. RESULTS: Hsp90beta was identified as a protein of the L-OA/AGRE-specific complex. Silencing p130(cas) and Hsp90beta significantly increased MMP-13 expression (about four- and twofold, respectively) and production. sip130(cas) affected to a lesser extent MMP-1 expression (twofold) and production. siNMP4 showed no effect. Expression of MMP-2, -3, -9 and -14 was unaffected. Silencing both Hsp90beta and p130(cas) had a significant additive effect on MMP-13, but not on MMP-1 expression, the level of which was similar to that with sip130(cas) alone. IL1beta decreased p130(cas) and Hsp90beta expression/production, indicating another pathway by which this cytokine upregulates MMP expression. The IL1beta-triggered signalling pathways responsible for MMP upregulation were unaffected in the silenced cells. CONCLUSION: This study illustrates the complex regulation of MMP-13 by showing the inhibitory effect of the two cytoplasmic molecules, p130(cas) and Hsp90beta, in L-OA chondrocytes.

[Interest of peri-operative immunonutrition in head and neck cancers]. / Intérêt de l'immunonutrition péri-opératoire en carcinologie cervico-faciale.

OBJECTIVE: Authors reported the results of a study on the application of immunonutrion in peri-operative (pre and postoperative) in head and neck cancer for all patients malnourished or not. In preoperative we used an oral treatmentand in postoperative an enteral one. MATERIALS AND METHODS: Prospective study concerning 78 patients (47 malnourished versus 31 not) having had heavy head and neck curative cancerology surgery. The mean follow up was of 10 months (from 7 to 16 month). They peri-operative immuno-enriched diet consisted, in pre-operative of 1000 kcal/j during 7 days of oral immunonutrition (Impact), and in post-operative, 1500 kcal/j during 10 days of enteral immunonutition (Crucial). The nutritional state was evaluated in pre-operative by simple clinical and biological parameters (size, weight, CMI "Corporal Mass Index", albumin, NRI "Nutritional Risk Index"), and in post-operative by the evolution of the weight and the CMI. The palatability of the product used in pre-operative and the patients' compliance to the treatment are studied using the satisfaction's multiple choice question paper. RESULTS: The study showed an improvement of the patients' nutritional and general state (regain appetite, less marked asthenia) and of the quality of life. The product used in preoperative was well tolerated, this oral supplementation led to the same beneficial effects of the enteral's. At eight days in preoperative, the average weight was 62.35 kg, the average CMI was 20.93, and the average NRI was 94.12. In post-operative the patients' nutritional state improved: at eight days, the average loss of weight was 2.82 kg, the average CMI was 22.2. At one and six months after respectively the average gain of weight was 2.17 kg and 6.11 kg, the average CMI was 23.71 and 25.16. The application of this protocol decreased the post-operative complications (13% reduction of the infectious complications and 6% diminution of the fistulas). The time of hospitalization is then reduced (1.7 days), and the life's longevity is improved. CONCLUSION: The results produced by this study, demonstrate the necessity to apply a peri-operative immuno-enriched diet systematically for all the patients with and without a degraded nutritional state, undergoing a heavy head and neck curative cancerology surgery.

Histone deacetylase inhibitors suppress interleukin-1beta-induced nitric oxide and prostaglandin E2 production in human chondrocytes.

OBJECTIVE: Overproduction of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) plays an important role in the pathogenesis of osteoarthritis (OA). In the present study, we determined the effect of trichostatin A (TSA) and butyric acid (BA), two histone deacetylase (HDAC) inhibitors, on NO and PGE(2) synthesis, inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 expression, and nuclear factor (NF)-kappaB DNA-binding activity, in interleukin-1beta (IL-1)-stimulated human OA chondrocytes, and on IL-1-induced proteoglycan degradation in cartilage explants. METHODS: Chondrocytes were stimulated with IL-1 in the absence or presence of increasing concentrations of TSA or BA. The production of NO and PGE(2) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of iNOS and COX-2 proteins and mRNAs was evaluated using Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Proteoglycan degradation was measured with dimethymethylene blue assay. Electrophoretic mobility shift assay (EMSA) was utilized to analyze the DNA-binding activity of NF-kappaB. RESULTS: HDAC inhibition with TSA or BA resulted in a dose-dependent inhibition of IL-1-induced NO and PGE(2) production. IL-17- and tumor necrosis factor-alpha (TNF-alpha)-induced NO and PGE(2) production was also inhibited by TSA and BA. This inhibition correlated with the suppression of iNOS and COX-2 protein and mRNA expression. TSA and BA also prevented IL-1-induced proteoglycan release from cartilage explants. Finally, we demonstrate that the DNA-binding activity of NF-kappaB, was induced by IL-1, but was not affected by treatment with HDAC inhibitors. CONCLUSIONS: These data indicate that HDAC inhibitors suppressed IL-1-induced NO and PGE(2) synthesis, iNOS and COX-2 expression, as well as proteoglycan degradation. The suppressive effect of HDAC inhibitors is not due to impaired DNA-binding activity of NF-kappaB. These findings also suggest that HDAC inhibitors may be of potential therapeutic value in the treatment of OA.

Randomised controlled trial comparing two methods of acetabular cup positioning during total hip arthroplasty.

BACKGROUND: Acetabular cup positioning is an important technical aspect in total hip arthroplasty. Most surgeons estimate cup abduction angle during surgery with the insertion rod position according to the patient's body anatomical landmarks or other reference points in the operating room. High acetabular component abduction angle is associated with an increased risk of dislocation, premature polyethylene wear and osteolysis. METHOD: To evaluate the potential benefits of a new technique for vertical acetabular cup positioning, 100 acetabular cups were randomised to be inserted with or without an inclinometer. Abduction angles were measured on postoperative radiographs by 2 evaluators blind to the treatment group. RESULTS: Of the cups, 57% (27/47) were positioned within the desirable abduction angle range of 40-49 with the inclinometer, compared with 50% (27/53) by visuospatial perception (p=0.454). The proportion of cups positioned outside a safe angle range of 30-55 was low in both groups: 6% (3/47) for the inclinometer group versus 4% (2/53) for the visuospatial perception group (p=0.536). CONCLUSION: The use of an inclinometer did not significantly improve the acetabular cup abduction angle obtained by our group of surgeons when compared with visuospatial perception. Newer techniques such as navigation may be useful in further optimising cup positioning and reducing the outliers.

Differential regulation of the bone morphogenic protein antagonist chordin in human normal and osteoarthritic chondrocytes.

OBJECTIVE: To investigate the distribution of the bone morphogenic protein (BMP) antagonist chordin in normal and osteoarthritic cartilage and synovial membranes, and its regulation in chondrocytes and synovial fibroblasts by inflammatory and growth factors. METHODS: Localisation of chordin in tissues was undertaken by immunohistochemistry and gene regulation was determined by real time polymerase chain reaction. RESULTS: In normal cartilage, chordin was found at low levels (mean (SD), 7.6 (1.3)%), mainly in the very superficial layers. In osteoarthritis, chordin was also found in the superficial layers (8.9 (1.1)%), though at a significantly higher level (24.7 (1.5)%) in the last two thirds of the cartilage. In contrast to normal cells, chordin mRNA and protein levels were significantly downregulated (p<0.01) in osteoarthritic chondrocytes by all the growth factors tested. Interferon gamma stimulated chordin expression in normal but not in osteoarthritic chondrocytes (p<0.0002), while interleukin 1 beta and tumour necrosis factor alpha did not affect the expression level. However, no difference was found in either the distribution or regulation of chordin in normal and osteoarthritic synovial membranes or synovial fibroblasts. CONCLUSIONS: The differential distribution and regulation of chordin in normal and osteoarthritic cartilage and chondrocytes suggests an involvement of this antagonist in the osteoarthritic process.

Interjoint coordination in lower limbs in patients with a rupture of the anterior cruciate ligament of the knee joint.

Previous studies of movement kinematics in patients with a ruptured anterior cruciate ligament (ACL) have focused on changes in angular displacement in a single joint, usually flexion/extension of the knee. In the present study, we investigated the effect of an ACL injury on the overall limb interjoint coordination. We asked healthy and chronic ACL-deficient male subjects to perform eight types of movements: forward squats, backward squats, sideways squats, squats on one leg, going up a step, going down a step, walking three steps, and stepping in place. Depending on the movement concerned, we applied principal component (PC) analysis to 3 or 4 degrees of freedom (DFs): thigh flexion/extension, knee flexion/extension, ankle flexion/extension, thigh abduction/adduction. The first three DFs were investigated in all movements. PC analysis identifies linear combinations of DFs. Movements with a fixed ratio between DFs are thus described by only one PC or synergy. PCs were computed for the entire movement as well as for the period of time when the foot was in contact with the ground. For both the control and the injured groups, two synergies (PC vectors) usually accounted for more than 95% of the DFs' angular excursions. It was possible to describe 95-99% of some movements using only one synergy. Compared to control subjects, injured subjects employed different synergies for going up a step, walking three steps, squatting sideways, and squatting forward, both in the injured and uninjured legs. Those movements may thus be more indicative of injury than other movements. Although ACL-deficiency did not increase asymmetry (angle between the PCs of the same movement performed on the right and the left sides), this result is not conclusive because of the comparatively low number of subjects who participated in the study. However, the finding that synergies in both legs of patients were different from those in control subjects for going up a step and walking three steps suggests that interjoint coordination was affected for both legs, so that the asymmetry index might have been preserved despite the injury. There was also a relationship between the asymmetry index for squatting on one leg, squatting forward, walking three steps and some of the outcomes of the knee injury and osteoarthritis outcome score (pain, symptoms, activities of daily living, sport and recreation function, and knee-related quality of life). This suggests that significant differences in the asymmetry index could be obtained if more severely-injured patients participated in this study. It is possible that subjects compensated for their mechanical deficiencies by modifying muscle activation patterns. Synergies were not only modified in injured subjects, but also rearranged: the percentage of movement explained by the first PC was different for the injured and/or uninjured legs of patients, as compared to the legs of the control group, for going up a step, going down a step, walking three steps, and squatting forward. We concluded that the analysis of interjoint coordination may be efficient in characterizing motor deficits in people with knee injuries.

Comparison of two methods for reconstruction of the posterior cruciate ligament using a computer based method: quantitative evaluation of laxity, three-dimensional kinematics and ligament deformation measurement in cadaver knees.

The aim of this paper is to present a biomechanical comparison of two different methods for reconstruction of the posterior cruciate ligament in cadaver knees. We used an original computer-based method allowing precise calculation of three-dimensional (3D) knee kinematic parameters as well as the estimation of combined graft deformation (elongation-flexion-torsion). After isolated posterior cruciate ligament (PCL) dissection, double bundle and 'over-the-bottom' methods were performed successively on each knee using synthetic polyester ligaments. The effect of pre-tensioning was tested with the 'over-the-bottom' method. antero-posterior (A-P) and rotational laxity as well as 3D kinematics were recorded and analysed. Our computer based method allowed us to show that both reconstruction methods were equivalent in restoring A-P and rotational laxity as well as kinematic curves. Combined deformation of the prostheses was equivalent for both ligaments.

Can altered production of interleukin-1beta, interleukin-6, transforming growth factor-beta and prostaglandin E(2) by isolated human subchondral osteoblasts identify two subgroups of osteoarthritic patients.

OBJECTIVE: To determine the capacity of human subchondral osteoarthritic osteoblasts (Ob) to produce interleukin (IL)-1beta, IL-6, transforming growth factor-beta (TGF-beta) and prostaglandin E(2) (PGE(2)), and determine if a relationship exists between IL-1beta, TGF-beta, PGE(2) and IL-6 production. METHODS: We measured the abundance of IL-1beta, IL-6, TGF-beta and PGE(2) using very sensitive ELISA in conditioned-media of human primary subchondral Ob from normal individuals and osteoarthritic patients. Selective inhibition of IL-6 or IL-6 receptor signaling was performed to determine its effect on PGE(2) production whereas the inhibiton of PGE(2) production was performed to determine its effect on IL-6 production. The expression of bone cell markers and urokinase plasminogen activator (uPA) activity was also determined. RESULTS: Osteoarthritic Ob produced all these factors with greater variability than normal cells. Interestingly, the production of IL-6 and PGE(2) by osteoarthritic Ob separated patients into two subgroups, those whose Ob produced levels comparable to normal (low producers) and those whose Ob produced higher levels (high producers). In those cells classified as high osteoarthritic Ob, PGE(2) and IL-6 levels were increased two- to three-fold and five- to six-fold, respectively, compared with normal. In contrast, while using their IL-6 and PGE(2) production to separate osteoarthritic Ob into low and high producers, we found that IL-1beta levels were similar in normal and all osteoarthritic Ob. Using the same criteria, TGF-beta levels were increased in all osteoarthritic Ob compared with normal. Reducing PGE(2) synthesis by Indomethacin [a cyclo-oxygenase (COX) -1 and -2 inhibitor] reduced IL-6 levels in all osteoarthritic Ob, whereas Naproxen (a more selective COX-2 inhbitor) reduced PGE(2) and IL-6 levels only in the high osteoarthritic group. Conversely, PGE(2) addition to osteoarthritic Ob enhanced IL-6 production in both groups. Moreover, the addition of parathyroid hormone also stimulated IL-6 production to similar normal levels in both osteoarthritic groups. In contrast, using an antibody against IL-6 or IL-6 receptors did not reduce PGE(2) levels in either group. The evaluation of alkaline phosphatase activity, osteocalcin release, collagen type I and uPA activity in osteoarthritic Ob failed to show any differences between these cells regardless to which subgroup they were assigned. CONCLUSIONS: These results indicate that IL-6 and PGE(2) production by subchondral Ob can discriminate two subgroups of osteoarthritic patients that cannot otherwise be separated by their expression of cell markers, and that endogenous PGE(2) levels influence IL-6 synthesis in osteoarthritic Ob.

A new generation of artificial ligaments in reconstruction of the anterior cruciate ligament. Two-year follow-up of a randomised trial.

We have undertaken a randomised clinical trial comparing two methods of reconstruction of the anterior cruciate ligament in patients with chronic instability. We used an ipsilateral bone-patellar-tendon-bone autograft in 27 patients and the Ligament Advancement Reinforcement System (LARS) artificial ligament in 26. Assessment before and at two, six, 12 and 24 months after surgery, included the history, physical examination, a modified International Knee Documentation Committee (IKDC) score, the Tegner score, the Knee Injury and Osteoarthritis Outcome Score (KOOS) and instrumented laxity testing. There were no cases of reactive synovitis or of infection of the knee, and there was no difference regarding the failure rate between the two groups. The IKDC showed no significant differences between the two groups at any stage of the follow-up. The KOOS evaluation showed consistently better results in all subscales for the LARS group during the first year of follow-up. After 24 months these differences were no longer evident. Instrument-tested laxity was greater in the LARS group at all stages of follow-up, but the differences were not significant at 24 months. Our findings suggest that at follow-up at 24 months the LARS ligament seems to be a satisfactory treatment option, especially when an early return to high levels of activity is demanded.

[Standards, options and recommendations: Good clinical practice in the dietetic management of cancer patients: hospital catering]. / Standards, options et recommandations: Bonnes practiques de diététique en cancérologie: la prestation alimentaire.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, involves a collaboration between the Federation of the French Cancer Centres (FNCLCC), the 20 French Regional Cancer Centres, some French public university and general hospitals and private Clinics and medical scientific societies. Its main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on a literature review followed by a critical appraisal by a multidisciplinary group of experts to produce the draft guidelines which are then validated by specialists in cancer care delivery. OBJECTIVES: To develop clinical practice guidelines for hospital catering for cancer patient using the methodology developed by the Standards, Options and Recommendations project. METHODS: Data were identified by a literature search of Medline and the reference lists of experts in the groups. After the guidelines were drafted, they were validated by independent reviewers. RESULTS: The main recommendations are: 1) While taking into consideration the specific needs of cancer patients, the dietician is responsible for the hygiene, the sanitary quality of alimentation, the equilibrium and nutritional quality of the hospital catering. 2) Ordering and distribution of meals, and clearing up afterwards contribute to the quality of hospital catering and the personnel who do this should have time and be willing to listen to the patients. 3) The ordering of meals should be adapted to individual patient's requirements and must take into account the patient's medication. 4) The method of transporting the food chosen by the institution (cold or warm method) should be respected. The personnel responsible should receive regular and specific training to use the method correctly. 5) The intake of patients with nutritional follow-up should be reliably and reproducibly evaluated by the personnel after every meal. 6) Patient satisfaction should be assessed once a year and the results of this assessment used to improve the quality of hospital catering. 7) The dietician is the interface between the accounts department, the medical wards, the hospital catering department and the patients.

Transforming growth factor-beta induced collagenase-3 production in human osteoarthritic chondrocytes is triggered by Smad proteins: cooperation between activator protein-1 and PEA-3 binding sites.

OBJECTIVE: To examine the signaling pathways leading to transforming growth factor-beta (TGF-beta) induced collagenase-3 production in human osteoarthritic (OA) chondrocytes, as well as the transcription factors and their binding sites involved in the transcriptional control of collagenase-3 gene. METHODS: Identification of the TGF-beta signaling pathway was by Western immunoblotting using specific antibodies for the phosphorylated forms of p44/42 and p38 MAPK, SAPK/JNK, and the Smad2 protein. Electromobility shift assays (EMSA) were carried out for activator protein- (AP-1), polyomavirus enhancer A (PEA-3), activin-response-element-like, Smad-binding-element-like, and TGF-beta inhibitory element oligonucleotides. Supershift assays using antibodies to the Jun, Fos, and Smad families of proteins were used for identification of transcription factors. Chondrocyte transfections were also performed using the -133CAT collagenase-3 promoter plasmid (containing PEA-3, AP-1, and TATA sites) and mutated AP-1 and PEA-3 sites. RESULTS: The primary target of TGF-beta induced collagenase-3 in OA chondrocytes was the Smad2 protein, with significant phosphorylation within 5 min. Contrasting with the Smad2, the untreated OA chondrocytes already had detectable levels of the phosphorylated forms of p38 and p44/42 MAPK. Of the oligonucleotides tested, EMSA revealed that TGF-beta treated OA chondrocyte proteins bound only to the AP-1 and PEA-3. Supershifts with the AP-1 oligonucleotide showed the presence of the Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) proteins in the untreated and TGF-beta treated OA chondrocytes, whereas only Smad proteins (Smad2, 3, 4) were present in the AP-1 binding proteins from the TGF-beta treated chondrocytes. The AP-1 mutation decreased both basal (95%) and TGF-beta induced (99%) collagenase-3 production, whereas the PEA-3 mutation decreased the basal (15%) but more significantly (50%) the TGF-beta induced transcription. CONCLUSION: Smad proteins are the main cytoplasmic signaling pathways in TGF-beta stimulated collagenase-3 in OA chondrocytes. The AP-1 site appears critical for upregulation of collagenase-3 production, but TGF-beta stimulation requires both AP-1 and PEA-3 sites for optimal response.

Correlation between patients' satisfaction and objective measurement of knee stability after ACL reconstruction using a patellar tendon autograft.

We evaluated the relationship between patients' satisfaction and objective measurements of knee stability after reconstruction of the ACL using a patellar tendon autograft. An examination of 59 patients 2-7 years after surgery was carried out. Assessment was made by the Knee and Osteoarthritis Outcome Score for patient satisfaction, a modified International Knee Documentation Committee form for clinical knee stability and a Telos stress radiography for PA stability. The results show that patients' satisfaction was much greater than the objective evaluation would suggest. We conclude that documenting mechanical knee stability alone is inadequate for follow-up studies and a questionnaire assessing patient satisfaction should be added.

Patient satisfaction needs as related to knee stability and objective findings after ACL reconstruction using the LARS artificial ligament.

The purposes of this study are to compare patient satisfaction with the objective measurement of knee stability and assess early complications following ACL reconstruction using a LARS artificial ligament. Forty-seven patients were reviewed 8-45 months after surgery. Assessment was made by the Knee and Osteoarthritis Outcome Score for patient satisfaction, a modified International Knee Documentation Committee form for clinical knee stability, and a Telos stress radiography for PA stability. Complications were assessed at interview and were double-checked with charts. The LARS artificial ligament may be a safe device to reconstruct an ACL tear. Documenting mechanical stability of the knee is inadequate when reporting follow-up studies and a questionnaire assessing patient satisfaction should be added to provide a better picture of the outcome and results.

Quality of life and home enteral tube feeding: a French prospective study in patients with head and neck or oesophageal cancer.

A prospective study was conducted to evaluate the impact of home enteral tube feeding on quality of life in 39 consecutive patients treated for head and neck or oesophageal cancer at the Centre François Baclesse in Caen, France. Patients were taken as their own controls. Quality of life was evaluated using the EORTC QLQ-C30 core questionnaire, and the EORTC H&N35 and OES24 specific questionnaires. The feeding technique tolerance was evaluated using a questionnaire specifically developed for this study. Two evaluations were made, the first a week after hospital discharge (n = 39) and the second 3 weeks later (n = 30). Overall, the global health status/quality of life scale score slightly improved; among symptoms, scale scores that significantly improved (P < 0.05) concerned constipation, coughing, social functioning and body image/sexuality. The physical feeding technique tolerance was acceptable while the technique was psychologically less tolerated with two-thirds of the patients longing to have the tube removed. One third of the patients was also uncomfortable about their body image. Home enteral tube feeding was responsible for not visiting family or close relations in 15% of patients, and not going out in public in 23%. We conclude that home enteral tube feeding is a physically well accepted technique although a substantial proportion of patients may experience psychosocial distress.

[Good clinical practice in the dietetic management of cancer patients]. / Bonnes pratiques pour la prise en charge diététique en cancérologie: la consultation.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of the French Cancer Centres (FNCL CC), the 20 French Cancer Centres and specialists from French Public Universities, General Hospitals and Private Clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feed-back from specialists in cancer care delivery. OBJECTIVES: To develop clinical practice guidelines according to the definitions of Standards, Options and Recommendations for the dietetic consultation for cancer patient. METHODS: Data have been identified by literature search wing Medline and the expert groups personal reference lists. Once the guidelines were defined, the document was submitted for review to 74 independent reviewers, and to the medical committees of the 20 French Cancer Centres. RESULTS: The main recommendations for the referral of cancer patients for dietary advice are: I) in oncology, there are 3 types of dietetic consultation: diagnostic, preventive and therapeutic; 2) the following cancer patients must have a dietetic consultation: i) those with, or at risk of malnutrition, ii) those without malnutrition but in need of counseling and iii) those at risk of treatment-related nutritional side effects; 3) a nutritional assessment is standard at the time of the first dietetic consultation. Patients must be given individualized and written advice; 4) the dietetic opinion and advice should be brought to the attention of medical staff to facilitate a multidisciplinary approach to cancer treatment; 5) patient's relatives should be involved in the dietetic management; 6) the efficacy of dietetic advice can be assessed by monitoring weight, gastrointestinal signs and patient satisfaction.

A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech.

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.

Abnormal regulation of urokinase plasminogen activator by insulin-like growth factor 1 in human osteoarthritic subchondral osteoblasts.

OBJECTIVE: Subchondral bone sclerosis is a common feature of osteoarthritis (OA), but the mechanisms responsible for this condition remain unresolved. We investigated the role of insulin-like growth factor 1 (IGF-1) and urokinase plasminogen activator (uPA) in human osteoblasts from subchondral bone obtained from the tibial plateaus of OA patients and normal individuals. METHODS: Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients at surgery and from normal individuals at autopsy. Levels of uPA and PA inhibitor 1 (PAI-1) levels were determined under basal conditions and after IGF-1 stimulation in conditioned media from osteoblasts by enzyme-linked immunosorbent assay. The activity of uPA was evaluated by specific substrate hydrolysis and zymography under basal conditions and after plasminogen stimulation, in the presence and absence of added IGF-1. Plasmin activity was also evaluated by specific substrate hydrolysis. RESULTS: Levels of uPA released by OA osteoblasts were significantly higher than normal. Addition of IGF-1 to osteoblasts significantly reduced uPA protein levels only in OA patients (P < 0.05). In contrast, the addition of uPA to osteoblasts did not modify IGF-1 levels in either normal or OA osteoblasts. Basal uPA activity was higher in OA than in normal osteoblasts. Interestingly, IGF-1 enhanced basal uPA activity in OA specimens in a dose-dependent manner. Addition of plasminogen promoted uPA activity in both normal and OA osteoblasts via a positive feedback loop due to plasmin generation, since this activity was inhibited by both PAI-1 and alpha2-antiplasmin. Unexpectedly, incubation with IGF-1 inhibited this positive feedback of plasminogen-dependent uPA activity in OA osteoblasts, but not in normal osteoblasts, in a dose-dependent manner. Hence, normal osteoblasts were relatively insensitive to IGF-1, whereas the same treatment reduced both uPA levels and plasminogen-dependent uPA activity in OA osteoblasts while it increased basal uPA activity in OA osteoblasts. This could not be explained by PAI-1 protein levels, which were similar in normal and OA osteoblasts in the presence and absence of IGF-1. IGF-1 also reduced plasmin activity in OA osteoblasts while it did not modify this activity in normal osteoblasts. CONCLUSION: These results suggest that in OA osteoblasts, the uPA/plasmin system functions normally, yet IGF-1 inhibits the positive feedback of plasmin on uPA activity. This inhibition may contribute to abnormal IGF-1- and uPA-dependent bone remodeling, ultimately leading to abnormal bone sclerosis in OA.

Cloning and expression of acetylcholinesterase from Bungarus fasciatus venom. A new type of cooh-terminal domain; involvement of a positively charged residue in the peripheral site.

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms.

We analyzed the production of Torpedo marmorata acetylcholinesterase (AChE) in transfected COS cells. We report that the presence of an aspartic acid at position 397, homologous to that observed in other cholinesterases and related enzymes (Krejci, E., N. Duval, A. Chatonnet, P. Vincens, and J. Massoulié. 1991. Proc. Natl. Acad. Sci. USA. 88:6647-6651), is necessary for catalytic activity. The presence of an asparagine in the previously reported cDNA sequence (Sikorav, J.L., E. Krejci, and J. Massoulié. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1865-1873) was most likely due to a cloning error (codon AAC instead of GAC). We expressed the T and H subunits of Torpedo AChE, which differ in their COOH-terminal region and correspond respectively to the collagen-tailed asymmetric forms and to glycophosphatidylinositol-anchored dimers of Torpedo electric organs, as well as a truncated T subunit (T delta), lacking most of the COOH-terminal peptide. The transfected cells synthesized similar amounts of AChE immunoreactive protein at 37 degrees and 27 degrees C. However AChE activity was only produced at 27 degrees C and, even at this temperature, only a small proportion of the protein was active. We analyzed the molecular forms of active AChE produced at 27 degrees C. The H polypeptides generated glycophosphatidylinositol-anchored dimers, resembling the corresponding natural AChE form. The cells also released non-amphiphilic dimers G2na. The T polypeptides generated a series of active forms which are not produced in Torpedo electric organs: G1a, G2a, G4a, and G4na cellular forms and G2a and G4na secreted forms. The amphiphilic forms appeared to correspond to type II forms (Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:776-785; Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:786-794), which are abundant in the nervous tissue and muscles of higher vertebrates (Bon, S., T. L. Rosenberry, and J. Massoulié. 1991. Cell. Mol. Neurobiol. 11:157-172). The H and T catalytic subunits are thus sufficient to account for all types of known AChE forms. The truncated T delta subunit yielded only non-amphiphilic monomers, demonstrating the importance of the T COOH-terminal peptide in the formation of oligomers, and in the hydrophobic character of type II forms.