Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.     

Urinary stress incontinence. Comparison of the Raz technique and the Bologna technique. Analysis of a series of 188 patients

OBJECTIVE: Our purpose was to evaluate and compare the perioperative morbidity and the long-term results of the Raz colposuspension and the Bologna operation for the treatment of stress urinary incontinence in women. STUDY DESIGN: Data of 188 women who underwent either a Bologna operation (group 1) or a Raz colposuspension (group 2) for the treatment of stress urinary incontinence between January 1, 1987 and December 1, 1995 were analysed retrospectively regarding history, preoperative evaluation, associated surgical procedures, complications and cure and failure rate. POPULATION AND METHODS: 80 patients were treated with the Bologna operation and 108 with the Raz colposuspension. Patients with prior anti-incontinence surgery, and patients with a preoperative diagnosis of urge incontinence were excluded from the study. Success was defined both subjectively (complete absence of complaint of stress urinary incontinence) and objectively (no evidence of loss of urine on cough provocation during physical examination). Survival curves were generated in each group for time to event data: "time to recurrent stress incontinence" and compared by the logrank test. A multivariate analysis using a Cox proportional hazards regression model was performed to indentify possible outcome predictors. RESULTS: The success rate of group 1 was significantly higher than that of group 2 (p = 0.00001). The median of success was 51 months in group 1 and 21 months in group 2 (p = 0.00001). The incidence of intraoperative complications in each group (inadvertent cystostomy, hemorrhage) did not differ. The multivariate analyses using the Cox regression model showed that the most highly correlated variable with the surgical cure rate was the type of surgical anti-incontinence procedure adopted: Bologna or Raz (p = 0.00001) CONCLUSION: In our hands, the Bologna operation has a higher cure rate than the Raz colposuspension. For us, when treating patients with stress urinary incontinence by the vaginal route, the Bologna operation is indicated when sufficient anterior vaginal tissue is available to create vaginal bands.     

Biochemical and pharmacological properties of SANORG 34006, a potent and long-acting synthetic pentasaccharide

SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the "synthetic pentasaccharide" (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 +/- 0.3 v 48 +/- 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 +/- 15 anti-factor Xa U/mg v 850 +/- 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti-factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti-factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50 values = 40.0 +/- 3.4 and 105.0 +/- 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti-factor Xa activity. SANORG 34006 enhanced rt-PA-induced thrombolysis and inhibited accretion of 125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.     

Ecstasy--cool dope with long-lasting effects?

The amphetamine derivative MDMA (3,4-methylenedioxymethamphetamine) was first synthesised in 1914 as an appetite suppressant, but was never used as such. MDMA is commonly known as "ecstasy" and has become a popular recreational drug of abuse at dance-clubs and rave parties, where it is combined with all-night dancing, crowded conditions, poor hydration and loud sound. This combination is probably the main reason why we have seen an upsurge in toxicity problems at rave parties, since all these factors are thought to promote or increase the toxicity of MDMA. The desired effects of MDMA are euphoria, increased energy and enhanced communication with others. Adverse effects are hyperthermia, rhabdomyolysis, acute renal failure, hepatotoxicity, depression and psychosis.     

Detection and preliminary characterization of matrix metalloproteinase activity in temporomandibular joint lavage fluid

MATERIALS AND METHODS: In the period 1986-1994, 2950 patients with cardiovascular diseases were surgically treated. In 2104 cases we placed biological or synthetic grafts to maintain vascular continuity. The most common has turned out to be abdominal aortic aneurysm. We treated 783 cases in emergency conditions. Staging and localization of infection has been the first aim in patients with synthetic vascular grafts. We studied signs and symptoms related to infections. In all cases we discovered the microorganism responsible of infection we started antibiotic therapy. RESULTS: Surgical infection incidence is 4.9% (154 cases). Series analysis has evidenced a decrease in infection incidence in the period 1986-1994. The most frequent infections are: the urinary tract infection (59 cases, 38.5%) followed by surgical wound infection (37 cases, 24.1%), respiratory tract infection (27 cases, 17.5%), vascular graft infection (23 cases, 14.4%). All patients underwent a preoperative antibiotic prophylaxis with 2 degrees-3 degrees generation cephalosporines. We noted a higher graft infection incidence in patients treated with aortobifemoral reconstruction. We handled surgical infection following two main directions: 1-antibiotic therapy, 2-surgical treatment and antibiotic therapy. CONCLUSIONS: We noted surgical technique improvement and correct application of an antibiotic prophylaxis form has turned out to be the "gold standard" in order to reduce cardiovascular surgical infections. To reduce sepsis or graft infection we can work on either of the following: 1) antibiotic therapy; 2) operative time reduction; 3) try to limit vascular surgery in case of concomitant gastrointestinal surgical disease; 4) using alloplastic vascular grafts with high biological compliance; 5) patency time reduction of invasive diagnostic technique.     

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.     

Oncocytic cancers of the thyroid. Hürthle cell cancers

Nonvalvular atrial fibrillation is a frequent finding in elderly patients; the risk of thromboembolic complications is comparable to that reported in patients with rheumatic atrial fibrillation. Recently, 6 multicenter clinical trials (5 primary prevention, 1 secondary prevention trail) have been published which demonstrate equivocally that oral anticoagulation therapy significantly reduces the embolic risk in patients with nonvalvular atrial fibrillation by about 67% to 87%. The target INR of anticoagulation with warfarin in 2 of these trials was 1.4 to 2.8 ("low-dose" warfarin); interestingly, the magnitude of risk reduction was similar to these 2 studies with "low-dose" warfarin as it has been reported by the others using full-dose warfarin with an INR target between 2.0 and 4.5. Side effects of oral anticoagulation (severe bleeding complications) were low in these trials. Thus, the benefit-risk ratio in these 6 clinical trials encourage the use of oral anticoagulation in patients with nonvalvular atrial fibrillation. It will be a challenge to transfer these results to clinical practice, and to define in more detail the risk-benefit ratios for subgroups of patients with atrial fibrillation, e.g. patients > 75 years of age, or patients with "lone" or paroxysmal atrial fibrillation. It is well established that patients with chronic atrial fibrillation undergoing medical or DC-cardioversion are at risk for thromboembolic complications. In previous studies, this risk appears to be in the range of 2% without concomitant anticoagulation, but only 0.33% in those patients with concomitant anticoagulation. Thus, it is widely accepted that patients should be anticoagulated for at least 2 weeks prior and after planned cardioversion. Recently, an alternative concept has been proposed omitting anticoagulation before cardioversion; instead, transesophageal echocardiography is used to exclude intracardiac thrombi. Because it is known that mechanical function of the left atrium and appendage is still impaired after cardioversion, this concept of echocardiographic-guided cardioversion does not assign the necessity of anticoagulation at the day of cardioversion, and 2 weeks afterwards. The safety aspects of this concept of echocardiographic-guided cardioversion is under current investigation.     

Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold

Reproducible and optimized complex formation between polyanionic DNA and a polycationic vector is a key aspect of nonviral gene transfer systems. To this end, several factors relevant to in vivo delivery have been tested repeatedly on several cell types. Gene transfer with a lipopolyamine (transfectam) in the presence of serum was increased over 10-fold by sequential addition of the lipid to DNA. Paradoxically, high complex concentrations (> 200 micrograms DNA/ml) led to large enhancements too, which points to the fact that formation of productive complexes is a slow process. Each parameter, more than compensates for the decreased efficiency generally observed with nonviral vectors in serum. Transfectam and PEI (polyethylenimine)-mediated transfection also improved after mild centrifugation of the complexes on to the cells. These individual factors were shown to be essentially multiplicative, leading altogether to approximately a 1000-fold transfection increase with a luciferase reporter gene. Finally, 25 cell lines and primary cells (including fibroblasts, hepatocytes and endothelial cells) were successfully transfected over a five orders-of-magnitude efficiency range, From this large set of data, a general relation between the overall transfection level (as measured by luciferase reporter gene expression) and the fraction of transfected cells (histochemically stained for beta-galactosidase) could be inferred. Finally, transfectam and PEI displayed similar trends over this large range of efficiencies, which reinforces the hypothesis of a common transfection mechanism where the key endosome-releasing stop occurs through a "proton sponge' effect.     

Therapeutic strategies in acute myocardial infarction. Results of STIM 93 registry

A registry was set up by the national college of cardiologists practicing in general hospitals in February 1993. The data concerned mode of admission, demographic details, initial clinical and haemodynamic evaluation and hospital outcome. Special attention was given to the electrocardiographic changes before and, in patients receiving thrombolytic therapy, after treatment. An analysis of predictive factors for mortality was performed both in the group of patients "revascularised" and in the group treated conventionally. One thousand and twenty three cases from 327 centres were analysed. There were 1292 men and 531 women, with an average age of 67.9 years. The average time interval from onset of symptoms to hospital admission was 5 h 30 min, 56.8% of patients arriving within 6 hours. 36.4% of the population underwent thrombolysis or angioplasty, 75% of patients under 75 years of age admitted before the 5th hours underwent a procedure of myocardial revascularisation. The hospital mortality was 14%, 8.7% in those revascularised and 17% in patients treated conventionally. Factors predictive of mortality were age, female gender, Killip Classes III or IV, systolic blood pressure of less than 100 mmHg, heart rate of more than 100/min and contraindications of thrombolysis. The maximum ST depression, the sum of ST depression, the sum of ST elevation and depression, were also significant predictive factors of a fatal hospital outcome in the whole population group and in patients treated conventionally. In the reperfused group, only the initial sum of ST elevation and depression was predictive of mortality: 120 minutes after the beginning of thrombolysis, the sum of ST elevations and of elevations and depressions was predictive of twice the mortality when the values exceeded 0.6 mv and 1.4 mv respectively.     

Clinical, morphologic and immunophenotypic data based on 10 cases of canine muco-cutaneous epidermotropic T-lymphoma (analogous to Mycosis Funcgo?de). Important of an animal model of spontaneous pathology

Our serie of ten canine cutaneous epitheliotropic T-cell lymphomas (CTCL), is found in old dogs, belonging mainly to the Boxer breed. Site on the mucous membranes (especially buccal), the muco-cutaneous junctions, their clinical expression is polymorphous. Lesions, follow on one after another (erythema, plaques, nodules) and are diversely associated in a given animal, the borders between the different stages often being difficult to establish. Adenopathies noted at the time of the diagnosis or during the course of the condition are accompanied by an involvement of the blood and organs (analogous to Sézary's disease). The progression of the disease can be very rapid in the buccal forms, which are generally aggressive, and in cases of violent, uncontrollable pruritus, which may be disturbing for the owner (with requests for euthanasia). The neoplastic infiltrate is constituted of small lymphocytes with hyperchromatic, convoluted nuclei (incipient stages), then large cells with a "histiocytic" appearance for the nodules. Epitheliotropism, which is maximal for the infiltrated plaque stage, shows up either in the form of a flux of totally epitheliotropic isolated cells (Ketron-Goodman type) or in that of Pautrier abscess-like collections. THe veterinary literature is in agreement that the CTCL cell expresses CD3, but two recent studies are in contradiction as regards its membership of helper or cytotoxic/suppressor populations. For our 10 cases, all the cells of lymphocytic morphology were, without exception, CD3+ and CD45+, irrespective of their situation within the epithelium or the chorion. The CD3+ cells in the epithelium were systematically CD8+, CD4- (confirming P.F. Moore's observations), expressing CD5 in a variable way, and, mostly, the Ki-67 nuclear proliferation Ag. The CD3+ cells of the chorion were exclusively, or mainly, CD8+, and occasionally CD4+. They expressed CD5 in a variable way, and, for a minority, the Ki-67 nuclear proliferation Ag. On the pathogenic level, it may be suggested that a T clone, CD8+, undergoes the "homing" phenomenon within the epithelium, enters the cell cycle, then manifests a tropism towards the chorion, which it infiltrates. Despite some particularities, which may be clinical (serious mucous attacks), cytological (the "histiocytic" appearance of the nodule cells) or immunophenotypic (expression of CD8, similar to what is observed in man in a considerable number of Pagetoid reticulosis), CTCL constitutes an interesting model of spontaneous pathology, and could prove useful in: - identifying various etiological factors (given that the dog, as a close commensal of man, is subject to the same environmental factors).