Aldehyde and phosphinate analogs of glutathione and glutathionylspermidine: potent, selective binding inhibitors of the E. coli bifunctional glutathionylspermidine synthetase/amidase

INTRODUCTION: The tripeptide glutathione is converted to glutathionylspermidine (Gsp) in Escherichia coli and in trypanosomatid parasites by an ATP-cleaving Gsp synthetase activity. In parasites, an additional glutathionylation forms bis-(glutathionyl)-spermidine, trypanothione, believed to be the major surveillance thiol involved in oxidant defense mechanisms in trypanosomatid parasites. In E. coli, the Gsp synthetase is part of a bifunctional enzyme opposed by the hydrolytic Gsp amidase. RESULTS: Gsp amidase and Gsp synthetase activities of the bifunctional E. coli enzyme can be separately targeted by potent, selective slow-binding inhibitors that induce time-dependent inhibition. The inhibitor gamma-Glu-Ala-Gly.CHO most probably captures Cys59 and accumulates as the tetrahedral adduct in the amidase active site. Inhibitory Gsp phosphinate analogs are phosphorylated by ATP to yield phosphinophosphate analogs in the synthetase active site. Binding of phosphinophosphate in the Gsp synthetase active site potentiates the inhibition affinity for the aldehyde at the Gsp amidase active site by two orders of magnitude. CONCLUSIONS: Time-dependent inhibition of the Gsp amidase activity by the aldehyde substrate analog supports previous work that suggests glutathionyl acyl-enzyme intermediate formation in the Gsp amidase reaction mechanism. Use of potent selective inhibitors against Gsp amidase (aldehyde) and Gsp synthetase (phosphinate) activities provides further evidence of interdomain communication in the bifunctional enzyme from E. coli.     

The endothelium-dependent response of human hepatic artery after preservation with the UW solution

The University of Wisconsin's (UW) solution has been used commonly for current liver transplantation. However, its effect on the vascular endothelium remains unclear. Experiments were designed to study the effects. Human hepatic arteries harvested from patients with hepatocellular carcinoma undergoing liver resection were preserved in 4 degree C physiological solution (group 1, the content showed on the text) and UW solution (group 2) for 1 hr. Segments of preserved and control (group 3) hepatic arteries were suspended in organ chamber to measure the isometric force. The relaxations to acetylcholine (ACH) and adenosine diphosphate in segments of hepatic artery with endothelium were significantly greater than those segments without endothelium. The maximal relaxation to ACH in arterial segments with endothelium of group 2 was significantly different from those of group 1 and 3 (group 1 to group 3, 82 +/- 2%, 57 +/- 6%, and 83 +/- 4% of the initial tension contracted by neoepinephrine (3 X 10-7 mole/l, P < 0.05). The maximal relaxation to adenosine diphosphate was similar to the response to ACH. Perfusate hypoxia (oxygen tension 30 +/- 5 mmHG) caused endothelium-dependent contraction of the arterial segments (group 1 to group 3, 233 +/- 32%, 276 +/- 35%, and 251 +/- 40% of the initial tension, P < 0.05). Endothelium-independent relaxation and contraction of human hepatic artery to sodium nitroprusside and norepinephrine were not altered by UW solution. In summary, the impaired endothelium-dependent relaxation by UW solution and prominent endothelium-dependent contraction to hypoxia of human hepatic artery would favor vasospasm and thrombus formation after liver transplantation.     

Functions of the transforming growth factor-beta superfamily in eyes

One human body is composed of 6 x 10(13) cells, and eyes are also composed of many cells of different functions. The cellular functions and intercellular interaction are regulated by many regulators including cytokines and growth factors to maintain the homeostasis. The transforming growth factor-beta (TGF-beta) superfamily, a large family of multifunctional factors, regulates various cellular functions, including cellular proliferation, migration, differentiation, apoptosis and extracellular matrix production. The TGF-beta superfamily contains about 30 multifunctional factors, and is divided into several families according to the sequence homology. The TGF-beta family, the activin family, and bone morphogenic proteins belong to the TGF-beta superfamily. TGF-beta superfamily members transduce signals through type I and type II serine/threonine type transmembrane receptors. The signals are transduced from receptors through nuclei by Smad family members, which are phosphorylated by the activated type I receptors and translocate from cytoplasm into nuclei. TGF-beta family members and the TGF-beta superfamily receptor family are expressed in ocular tissues including the cornea, ciliary epithelium, lens epithelium, retina, and blood vessels. This observation suggests the importance of the TGF-beta superfamily in eyes. Smad family members (Smad 1, Smad 2, Smad 3 and Smad 4) are expressed in the cultured retinal pigmant epithelial cell line (D407), in which TGF-beta and activin A stimulate the translocation of Smad 2, but not Smad 1 into nuclei, whereas bone morphogenetic protein (BMP) stimulates that of Smad 1, but not Smad 2. TGF-beta superfamily members play important roles in the pathogenesis of retinal neovascularization and in the wound healing process of corneal tissue. TGF-beta inhibits the endothelial functions, but, stimulates angiogenesis in vivo. TGF-beta is involved in the formation of abnormal connective tissue in corneal wound healing. In these processes, many cytokines and growth factors are involved, interacting with each other and forming networks. It is mandatory to clarify the networks to investigate molecular pathogenesis and new therapeutic agents.     

Ischemia-associated intraventricular conduction disturbances during exercise testing as a predictor of proximal left anterior descending coronary artery disease

To determine the incidence and significance of transient intraventricular conduction abnormalities occurring in association with myocardial ischemia during exercise testing, the recordings of 2,200 consecutive exercise tests were reviewed. Ten patients (0.45%) were identified as having both ischemia and intraventricular conduction abnormalities that developed transiently during the exercise test. In all 10 patients both typical angina and electrocardiographic evidence of ischemia developed during exercise. Among the 10 patients, left anterior hemiblock developed in 4, left posterior hemiblock in 2, right bundle branch block (RBBB) in 2, RBBB with left axis deviation in 1, and left anterior hemiblock progressing to complete left bundle branch block (LBBB) in 1. All 10 patients had cardiac catheterization showing significant obstruction of the left anterior descending (LAD) coronary artery at or before the origin of the first septal branch. Eight patients were treated surgically and 2 medically, all with relief of ischemic symptoms. Nine of the 10 had repeat exercise stress testing without angina or electrocardiographic evidence of ischemia and without recurrence of the transient intraventricular conduction disturbance. It is concluded that the development of transient intraventricular conduction abnormalities associated with myocardial ischemia during exercise testing is an uncommon occurrence (0.45%). When such conduction disturbances do develop, the existence of significant disease in the proximal portion of the LAD coronary artery is strongly suggested. With control of myocardial ischemia, the transient conduction disturbances during exercise are ameliorated.