Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205–316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu³⁰² residue into fragments 205–302 and 303–316. Upon incubation for 2–5 days of fragment 205–302 with a 5–fold excess of peptide 303–316, prepared by solid phase synthesis, with V8-protease in 0.1M ammonium acetate, pH6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to ñ90% (based on fragment 205–302). The same procedure was used to prepare also the thermolysin fragments 205–315 and 205–311 by enzymatic coupling of fragment 205–302 to peptide 303–315 or 303–311, these last prepared by proteolytic digestion of the synthetic peptide 303–316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205–316 of thermolysin at the level of its helical segment encompassing residues 301–312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Superselective embolization of bladder arteries in the treatment of intractable bladder haemorrhage

Bladder hemorrhage following radiation therapy is a serious complication in patients undergoing this treatment. Several methods have been proposed to control this particular situation; however, results have been far from satisfactory, with the exception of drastic measures such as hypogastric artery ligation and radical cystectomy. We recently used a method of superselective embolization of the bladder arteries which enabled us to control severe intractable bleeding in a patient submitted to bladder irradiation for a transitional cell infiltrating carcinoma. Compared to selective embolization and other methods, the advantages of superselective embolization are a lower recurrence rate concerning bleeding, fewer side-effects and the possibility of using adaptable embospheres (150-1000 micron) which, on account of their marked plasticity, offer better occlusion of the vessels. For these reasons, superselective embolization of the bladder arteries should be considered as the treatment of choice in intractable bladder hemorrhage.     

Arterial Shunt with Pump Infusion Line for the Treatment of Chronic Thoracic Aortic Aneurysms: The "Modified Shunt of Gott"

Dissection of the aneurysm is the most dangerous step during graft replacement of the descending thoracic aorta. Sudden hemorrhage may follow wall rupture or disruption of major collaterals before the aorta can be clamped. A simple modification of the classic Gott is illustrated, which makes the shunt work also as a partial bypass if needed, with rapid reinfusion of blood losses. Nineteen of 25 patients requiring resection of descending aortic aneurysms from 1982 to 1990 were treated with this method with no mortality.     

新生大鼠缺氧缺血后不同脑区神经元变性的动态观察

目的：观察新生大鼠缺氧缺血（HI）后不同脑区神经元变性的动态变化,探讨其与星形胶质细胞反应的关系。方法：采用Fluoro-Jade B（FJB）染色法及免疫组化法分别观察变性神经元荧光染色强度及胶质纤维酸性蛋白（GFAP）表达的动态变化。结果：正常新生大鼠各脑区内均未见FJB阳性细胞,GFAP呈弱表达。HI后1d即可见FJB阳性细胞,3d阳性细胞增多,5d达高峰,以纹状体受累最重,皮质次之,海马各区易损顺序为CA1、CA4、CA2、CA3、齿状回。GFAP表达的强度与FJB阳性细胞数密切相关。结论：新生大鼠HI后脑易损部位为纹状体、皮质、海马,FJB染色是一种标记变性神经元的有效方法,反应性胶质细胞增生是应答神经元损伤的结果。     

Spectroscopic investigations on dermorphin and its [L-Ala2] analog

Dermorphin, DM, (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) and its [l -Ala²]-analog, LDM, have been investigated in aqueous and DMSO solutions by means of ¹H- and ¹³C-n.m.r., CD and u.v. with the aim of establishing possible differences in the conformational preferences of the two peptides. A study of the pH dependence of ¹³C chemical shifts resulted in the determination of the pK_a of the α-amino group (6.97 0.02 for DM and 7.13 0.02 for LDM). A significantly higher value (7.75) is obtained for LDM from the pH dependence of CD spectra. This difference is attributed to the presence of associates at the high concentrations required for the ¹³C-n.m.r. measurements. The average pK_a's of the two phenol groups have also been obtained and the values determined from u.v. absorption spectra (9.79 for DM, 9.85 for LDM) are in fair agreement with those determined from the CD spectra (9.94 for DM and 10.11 for LDM). An analysis of titration shifts in ¹³C-n.m.r. spectra indicates that the α-amino group is not involved in interactions stabilizing folded conformers in aqueous solution. Temperature coefficients and exchange times of the NH protons suggest that both peptides exist in DMSO-d₆ as a mixture of rapidly interconverting conformers. A model is proposed in terms of an extended β-sheet conformation stabilized by intermolecular association at the high concentrations required for the n.m.r. measurements. CD spectra are dominated by contributions of the aromatic chromophores and cannot be used as a source of information about the main chain conformation. However, differences between the spectra of the two peptides indicate that the conformational freedom of one tyrosine side chain (probably Tyr¹) is slightly more restricted in DM than in LDM.     

Constrictive Epicarditis After Open Heart Surgery: The Turtle Cage Operation

A 60-year-old man developed constrictive epicarditis within 1 year after isolated mitral valve replacement (MVR). At reoperation, decortication of the thick epicardial layer resulted, impossible without a high risk of injury of the myocardium and major coronary arteries. Therefore, multiple longitudinal and transverse incisions were performed on the epicardial peel, which at the end acquired a turtle cage appearance allowing myocardial reexpansion, relief of constriction, and restoration of adequate hemodynamics.