A possible zymogen self-destruct mechanism preventing pancreatic autodigestion

Incubation at 37 degrees C of human cationic trypsinogen purified by PAGE electrophoresis, results in development of proteolytic activity (enzyme Y) capable of rapidly degrading cationic and anionic trypsinogens to inert products. Enzyme Y appears to be a serine protease with a molecular weight of about 20,000 daltons and is different from any of the known pancreatic enzymes. The active enzyme may be derived from trypsinogen itself or a hitherto unrecognized precursor contaminating the trypsinogen fraction used in this work. Appearance of enzyme Y activity seems to be associated with the presence of traces of free trypsin. Enzyme Y possesses insignificant or no activity when tested with a variety of synthetic trypsin, chymotrypsin and other protease substrates. It is not inactivated by the specific trypsin and chymotrypsin inhibitors TLCK and TPCK, but its activity is reduced gradually by increasing concentrations of pancreatic secretory trypsin inhibitor. Ca2+ concentrations greater than 3 mM strongly inhibit enzyme Y, and diisopropylfluorophosphate completely inactivates it. The enzyme is stable when incubated at pH 1.9 and 37 degrees C for 30 min and its activity is not abolished by treatment with Hg2+. When added to pancreatic juice with low inhibitor content it causes rapid inactivation of zymogens without significant release of active enzymes or reduction of pancreatic trypsin inhibitor. Its physiological role may be perceived as a second line of defense against premature intrapancreatic activation of zymogens. Enzyme Y activity may be generated when trypsin inhibitor, the first line of defense, is sufficiently depleted by complex formation with inappropriately released trypsin to permit dissociation of a small amount of trypsin from this complex. This in turn may lead to activation of enzyme Y and inactivation of the zymogens of pancreatic proteases.     

Herz- und Herz-Lungen-Transplantation

Orthotopic heart transplantation (HTX) is nowadays the worldwide accepted gold standard for the treatment of terminal heart failure. The main indications for HTX are non-ischemic dilatative (54%) and ischemic (37%) heart failure. In the acute phase after HTX the survival rate is approximately 90%. Good short and long-term results with survival rates ranging from 81% after 1 year to more than 50% after 11 years demonstrate that there is currently no real treatment alternative to HTX for treatment of end-stage heart failure. In the case of irreversible pulmonary hypertension in combination with end-stage heart failure or complex congenital heart syndromes, a combined heart and lung transplantation (HLTX) is necessary. Compared with HTX the short-term survival of HLTX is reduced, mostly for technical reasons. Improved long-term results after HTX and HLTX are a result of highly specialized transplantation units and effective immunosuppression. However, a major problem is the shortage of organ donors in Germany and the resulting long waiting times for patients with frequently occurring blood groups of up to 10 months for transplantation. The consequence of the latter is the ever increasing number of implanted cardiac assist devices in patients not only as a bridge to transplant but also as destination therapy.     

Shape matters in protein mobility within membranes

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation.Brownian motion plays an essential role in biological processes. Since the pioneering experiments of Perrin (1), the observation of diffusing objects has emerged as a mean to extract the rheological properties of the surrounding medium or the probe particle size. The theoretical investigation of diffusion of proteins within membranes has been studied widely going back to P. G. Saffman and M. Delbrück (SD). They investigated the hydrodynamic drag acting on a membrane inclusion when the membrane is described as a 2D fluid sheet of viscosity embedded within a less viscous fluid of viscosity η (2). In this theory, the diffusion coefficient D₀ in the limit of a large viscosity contrast between the membrane and bulk fluid is given by:The length is the length scale over which flow is generated within the bilayer by the inclusion, k_BT is the thermal energy, and γ is Euler’s constant. This model predicts a logarithmic dependence of D₀ on the protein radius a_p, which has been confirmed for some in vitro experiments on membranes containing transmembrane proteins (see ref. 3 and references therein). In contrast, the experiments of Gambin et al. (4) showed significant deviations from the SD theory.A possible origin for the discrepancy observed by Gambin et al. (4) is the significant local membrane deformation due to the interaction between the inclusion and the lipid bilayer (5). Naji et al. suggested in ref. 6 that inclusions experience additional dissipation, either due to internal flows within the membrane or to additional fluid flows produced by the deformed membrane. This work triggered a number of theoretical studies investigating the coupling of inclusion proteins with the membrane that had been pioneered by the Seifert’s group (see ref. 7 and references therein). Such studies have systematically gone beyond the SD model by including additional effects (8–12). So far, a thorough verification of these ideas has not been attempted. To investigate the effect of the protein–lipid coupling on the protein mobility, we study its dependence on membrane tension, because this parameter affects the local membrane deformation.In this work, we compare the mobility of two transmembrane proteins with the same lateral size, aquaporin 0 (AQP0) and a voltage-gated potassium channel (KvAP), reconstituted in giant unilamellar vesicles (GUVs). Whereas AQP0 does not deform locally the bilayer, KvAP locally bends the membrane (13). Using single-particle tracking (SPT), we demonstrate that the curvature-coupled protein KvAP undergoes a significant increase in mobility under tension, whereas the mobility of the curvature-neutral water channel AQP0 is insensitive to it. This difference, which goes beyond the SD model, is explained by an approach that includes the interplay between membrane deformation and friction with the surrounding medium and within the bilayer. This is compelling evidence that the Brownian motion of a shaping-membrane protein is not simply dependent on the inclusion size but also related to the lateral extension of the deformed membrane patch, which depends on tension.     

Application of the “Hybrid Approach” to Chronic Total Occlusion Interventions: A Detailed Procedural Analysis

Objective

To assess the outcomes of the “hybrid” approach to chronic total occlusion (CTO) percutaneous coronary interventions (PCIs).

Background

The “hybrid approach” to CTO PCI advocates appropriate and early change of crossing strategy to maximize success, safety, and efficiency.

Methods

We prospectively recorded and analyzed detailed step‐by‐step procedural data in 73 consecutive CTO PCI cases performed by a single operator between July 2011 and August 2012.

Results

Technical success was achieved in 66 of 73 cases (90.4%). Mean patient age was 65 ± 7 years, and 30% had prior coronary artery bypass surgery. Dual injection was used in 78%. The primary approach was retrograde in 9 cases (12.5%) and antegrade in 64 cases (87.5%), of whom 25 cases (39.1%) underwent retrograde attempt after failed antegrade approach. The initial crossing approach was successful in 40 cases (54.8%), but 32 cases (44%) required 3.6 ± 1.4 approach changes (range 2–7). Antegrade wire escalation, antegrade dissection/reentry, and retrograde crossing were utilized in 97.2%, 46.6%, and 46.6% of cases, respectively. Among successful cases, the final CTO crossing technique was antegrade wire escalation in 50.0%, antegrade dissection/reentry in 24.2%, and retrograde in 25.8%. The mean procedure time, fluoroscopy time, and air kerma radiation exposure until CTO crossing or stopping the procedure were 66 ± 55 minutes, 25 ± 23 minutes, and 2.3 ± 1.9 Gray, respectively. Three patients (4.1%) had a major complication.

Conclusion

In the “hybrid approach” to CTO PCI, changes in crossing strategy were needed in approximately half the cases, resulting in high success and low complication rates. (J Interven Cardiol 2014;27:36–43)

     

Malignant fibrous histiocytoma presenting with complete opacification of the hemithorax: A case report

BACKGROUND

Malignant fibrous histiocytoma, a subtype of primary lung sarcoma is a very rare disease. It usually presents as a lung nodules and the final diagnosis is made by immunohistochemical studies.

METHODS

A 45-year-old patient presented with progressive dyspnea, dry cough and right shoulder pain. Chest X-ray revealed complete opacification of the right hemithorax. Chest computed tomography confirmed the presence of a heterogeneous lesion occupying the whole right hemithorax causing a mass effect on the trachea. Ultrasound guided biopsy was done and final pathology was suggestive of malignant fibrous histiocytoma.

CONCLUSION

Progressive dyspnea in young otherwise healthy patients should be investigated early on. In our case the presence of right shoulder pain indicates advance disease illustrated by the singular imaging findings.     

Protein aggregation and prionopathies

Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are “transmissible” between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays.     

Lack of Amphetamine-Like Effects After Administration of Mefenorex in Normal Young Subjects

Mefenorex is an indirect sympathomimetic amine which acts as an anorectic drug and is used in combination with low diet to treat excess weight. The central nervous system (CNS) effects of mefenorex were assessed in a randomized, double-blind, three-way cross-over, placebo-controlled study involving nine healthy young male volunteers. They received either a single oral dose of mefenorex 80 mg (twice the recommended dose) or d-amphetamine sulfate 18 mg or a placebo at 1-week intervals. CNS pharmacodynamic measurements consisted of subjective evaluation (visual analogue scales and the Addiction Research Centre inventory (ARCI)), EEG, psychomotor performance and attention (tracking, simple and choice reaction times, tapping, continuous performance task, DSST, body sway) and memory (working memory and recall of a word list). d-Amphetamine produced a typical psychostimulant EEG profile (significant decrease in slow delta waves and increase in fast beta activities), significantly increased amphetamine, benzedrine and morphine–benzedrine scores of ARCI and significantly decreased body sway compared to placebo and mefenorex. A trend in favour of a stimulant effect occurred for all other parameters (particularly speed of reaction) and no changes of memory were noticed. In contrast, mefenorex did not produce an amphetamine-like EEG profile, neither significantly changed ARCI scores nor significantly modified psychomotor and memory performance compared to the placebo, although it induced a decrease in body sway. In conclusion, the present results indicate that a single oral dose of mefenorex, at twice the recommended daily dose, does not possess amphetamine-like subjective and EEG stimulant effects or sensations of well-being, often encountered with drugs of abuse liability potential, in a healthy young population.