Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.     

Differential action of proteases from Trimeresurus malabaricus, Naja naja and Daboia russellii venoms on hemostasis

The action of venom proteases and their role in hemostasis has been compared in the venoms of Trimeresurus malabaricus, Daboia russellii and Naja naja from the Southern region of Western Ghats, India. These venoms exhibit varying amounts of proteolytic activity and also influence hemostasis differently. Casein hydrolyzing activity of T. malabaricus venoms was 16 and 24 fold higher than those of N. naja and D. russellii venoms, respectively. With the synthetic substrate TAME, the highest activity was observed in T. malabaricus venom. N. naja venom did not hydrolyze TAME even at higher concentrations. These variations in proteolytic activity also influenced the coagulation process. T. malabaricus and D. russellii venoms are strongly procoagulant and reduce the re-calcification time from 148 to 14 and 12 s, respectively. Similarly, both T. malabaricus and D. russellii venoms reduce the prothrombin time from 12.5 to 6.0 s. On the other hand, N. naja venom is anticoagulant and prolongs re-calcification time to 600 s and prothrombin time to 42 s. In spite of varied effects on hemostasis, all the venoms hydrolyze fibrinogen. T. malabaricus venom hydrolyses both Aalpha and Bbeta subunits. While D. russellii and N. naja venoms hydrolyse only Aalpha. None of these venoms hydrolyze the gamma subunit of fibrinogen. Inhibition studies with specific protease inhibitors revealed that both N. naja and T. malabaricus venoms contain only metalloproteases. D. russellii venom contained both serine and metalloproteases. Only, T. malabaricus venom exhibited thrombin-like activity and induces fibrin clot formation with purified fibrinogen within 58 s. Even though D. russellii venom exhibits procoagulant activity, it did not show thrombin-like activity and may act on other coagulation factors.     

Characterization of a glutathione metabolic mutant of Mycobacterium tuberculosis and its resistance to glutathione and nitrosoglutathione

Glutathione is a tripeptide and antioxidant, synthesized at high levels by cells during the production of reactive oxygen and nitrogen intermediates. Glutathione also serves as a carrier molecule for nitric oxide in the form of S-nitrosoglutathione. Previous studies from this laboratory have shown that glutathione and S-nitrosoglutathione are directly toxic to mycobacteria. Glutathione is not transported into the cells as a tripeptide. Extracellular glutathione is converted to a dipeptide due to the action of transpeptidase, and the dipeptide is then transported into the bacterial cells. The processing of glutathione and S-nitrosoglutathione is brought about by the action of the enzyme gamma-glutamyl transpeptidase. The function of gamma-glutamyl transpeptidase is to cleave glutathione and S-nitrosoglutathione to the dipeptide (Cys-Gly), which is then transported into the bacterium by the multicomponent ABC transporter dipeptide permease. We have created a mutant strain of Mycobacterium tuberculosis lacking this metabolic enzyme. We investigated the sensitivity of this strain to glutathione and S-nitrosoglutathione compared to that of the wild-type bacteria. In addition, we examined the role of glutathione and/or S-nitrosoglutathione in controlling the growth of intracellular M. tuberculosis inside mouse macrophages.     

Abnormal mitochondrial dynamics and synaptic degeneration as early events in Alzheimer's disease: implications to mitochondria-targeted antioxidant therapeutics

Synaptic pathology and mitochondrial oxidative damage are early events in Alzheimer's disease (AD) progression. Loss of synapses and synaptic damage are the best correlates of cognitive deficits found in AD patients. Recent research on amyloid beta (Aβ) and mitochondria in AD revealed that Aβ accumulates in synapses and synaptic mitochondria, leading to abnormal mitochondrial dynamics and synaptic degeneration in AD neurons. Further, recent studies using live-cell imaging and primary neurons from amyloid beta precursor protein (AβPP) transgenic mice revealed reduced mitochondrial mass, defective axonal transport of mitochondria and synaptic degeneration, indicating that Aβ is responsible for mitochondrial and synaptic deficiencies. Tremendous progress has been made in studying antioxidant approaches in mouse models of AD and clinical trials of AD patients. This article highlights the recent developments made in Aβ-induced abnormal mitochondrial dynamics, defective mitochondrial biogenesis, impaired axonal transport and synaptic deficiencies in AD. This article also focuses on mitochondrial approaches in treating AD, and also discusses latest research on mitochondria-targeted antioxidants in AD. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Prostatic Cell-Specific Regulation of the Synthesis of MUC1-Associated Sialyl Lewis a

Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe^a) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe^a. We have found that SAHA enhances the production of sLe^a in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe^a is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe^a in RWPE-1 cells is resulted from upregulation of B3GALT1 gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe^a despite expressing high levels of B3GALT1. However, the MUC1-associated sLe^a is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe^a is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe^a. Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.     

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.     

Annexin I modulates cell functions by controlling intracellular calcium release.

Annexin I is an intracellular protein in search of a function. Ex vivo it has calcium- and phospholipid-binding properties. To evaluate its role in vivo, MCF-7 cells were stably transfected with annexin I in sense or antisense orientations. In cells overexpressing annexin I, calcium release was abrogated on stimulation of purinergic or bradykinin receptors, whereas non-transfected cells or cells with down-regulated annexin I released calcium within seconds. Basal calcium and calcium stores were not affected. The impaired calcium release was paralleled by a down-regulation of the activities of phospholipase C, group II phospholipase A2, and E-cadherin with altered adhesion and enhanced tumor growth on soft agar. Significantly smaller tumors, with the histologically most differentiated cells, were observed in nude mice inoculated with cells transfected with the antisense rather than with the sense plasmid. These observations indicate that annexin I modulates cell functions by controlling intracellular calcium release. Frey, B. M., Reber, B. F. X., Vishwanath, B. S., Escher, G., Frey, F. J. Annexin I modulates cell functions by controlling intracellular calcium release.