We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of the serpin reaction in which the acyl-enzyme intermediate must first be exposed to hydrolyzing water and aqueous deacylation. Substitution of the breach tryptophan in PAI-1 (Trp175), a residue found in virtually all known serpins, with phenylalanine altered the kinetics of the reaction mechanism and impeded the ability of PAI-1 to spontaneously become latent without compromising the inherent rate of cleaved loop insertion or partitioning between the final inhibited serpin-proteinase complex and hydrolyzed serpin. Kinetic dissection of the PAI-1 inhibitory mechanism using multiple target proteinases made possible the identification of a single rate-limiting intermediate step coupled to the molecular interactions within the breach region. This step involves the initial insertion of the proximal reactive center loop hinge residue(s) into beta-sheet A and facilitates translocation of the distal P'-side of the cleaved reactive center loop from the substrate cleft of the proteinase. Substitution of the tryptophan residue raised the kinetic barrier restricting the initial loop insertion event, significantly retarding the rate-limiting step in tPA reactions in which strong exosite interactions must be overcome for the reaction to proceed.

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.

Objectives:Sacral neuromodulation has gained increased worldwide acceptance as the standard of care in patients with refractory overactive bladder (OAB) and non-obstructive urinary retention (NOUR). This review will detail the evolution of the technology.Materials and Methods:The mechanism of action and advances in treatment, including tined lead, fluoroscopic imaging, and smaller implantable pulse generator (IPG) are reviewed. This discussion also explores expanding indications and future advances including interstitial cystitis, chronic pelvic pain, neurogenic bladder, fecal incontinence, constipation, and dysfunctional elimination syndrome in children.Results:Sacral neuromodulation (SNM) exerts its influence by modulation of sacral afferent inflow on storage and emptying reflexes. The tined lead allows for placement and stimulation to be performed in the outpatient setting under local anesthesia with mild sedation. Lead migration has been minimal and efficacy improved. The use of fluoroscopy has improved accuracy of lead placement and has led to renewed interest in bilateral percutaneous nerve evaluation (PNE). Bilateral PNE can be performed in the office setting under local anesthesia, making a trial of therapy less expensive and more attractive to patients. A smaller IPG has not only improved cosmesis, but decreased local discomfort and need for revision. The role for SNM continues to expand as clinical research identifies other applications for this therapy.Conclusions:Our understanding of SNM, as well as technological advances in therapy delivery, expands the pool of patients for which this form of therapy may prove beneficial. Less invasive instrumentation may even make this form of therapy appealing to patients without refractory symptoms.

osttraumatic pulmonary arteriovenous fistula (PAVF) is a rare clinical entity. Most PAVFs are congenital and arise in the setting of hereditary hemorrhagic telangiectasia. Acquired PAVFs are rare and have been reported with metastatic thyroid carcinoma, cirrhosis, pulmonary schistosomiasis, actinomycosis, mitral stenosis, Fanconi's syndrome, and trauma. We present a case of acquired PAVF presenting 1 year after a thoracic stab wound injury with a review of the previously reported cases and a discussion. CASE REPORTThe patient is a 27-year-old male who sustained a single stab wound to the right chest in the para-sternal region. His vital signs were stable on initial evaluation and he reported only shortness of breath. A right chest tube was placed with minimal return of blood. Computed tomography of the chest was performed, which revealed a large unevacuated right hemothorax. A second chest tube was placed, without improvement, based on serial chest films.The patient was taken to the operating room for right posterolateral thoracotomy and evacuation of clotted hemothorax. A large amount of clotted blood was evacuated and active chest wall bleeding was controlled with electrocautery. A pulmonary laceration with an associated 5-cm parenchymal hematoma was identified in the right upper lobe. The segment containing the laceration and the hematoma was resected with a single application of a gastrointestinal anastomosis-stapling device. The patient returned to the operating room the following day for significant, persistent bloody chest tube output and a drop in hematocrit to 18%. A right hemothorax was evacuated, but no site of bleeding was identified. The patient was extubated the following day and discharged to home on postoperative day 6.The patient was lost to follow up during the next several months. He presented to an outlying hospital with complaints of progressive weakness, fatigue, shortness of breath, and several episodes of syncope a year after initial surgery. On physical examination, the patient appeared anxious. Jugular venous distension was not present. The chest was clear to auscultation bilaterally and there was no appreciable murmur heard over the precordium. Resting O 2 saturation on room air was 85% and fell to 75% with exertion. Hemoglobin and hematocrit were 19.0 and 55%, respectively. A small right pleural effusion was noted on chest X-ray film. Computed tomography of the chest showed a mild amount of scarring in the right upper lobe and was negative for pulmonary embolism. A transesophageal echocardiogram was performed with a bubble study and this demonstrated a right to left shunt. A pulmonary angiogram was obtained, which revealed a PAVF with significant flow between the inferior division of the right pulmonary artery and the right superior pulmonary vein ( Figs. 1 and 2). Pulmonary function tests were performed and the FEV 1 (forced expiratory volume in 1 sec) was reported at 2.73 (Ͼ40% predicted value).The interventional radiologist ruled out therapeutic embolization based on the large size ...