Ligand selectivity of soluble guanylyl cyclase: effect of the hydrogen-bonding tyrosine in the distal heme pocket on binding of oxygen, nitric oxide, and carbon monoxide

Although soluble guanylyl cyclase (sGC) functions in an environment in which O(2), NO, and CO are potential ligands for its heme moiety, the enzyme displays a high affinity for only its physiological ligand, NO, but has a limited affinity for CO and no affinity for O(2). Recent studies of a truncated version of the sGC beta(1)-subunit containing the heme-binding domain (Boon, E. M., Huang, S H., and Marletta, M. A. (2005) Nat. Chem. Biol., 1, 53-59) showed that introduction of the hydrogen-bonding tyrosine into the distal heme pocket changes the ligand specificity of the heme moiety and results in an oxygen-binding sGC. The hypothesis that the absence of hydrogen-bonding residues in the distal heme pocket is sufficient to provide oxygen discrimination by sGC was put forward. We tested this hypothesis in a context of a complete sGC heterodimer containing both the intact alpha(1)- and beta(1)-subunits. We found that the I145Y substitution in the full-length beta-subunit of the sGC heterodimer did not produce an oxygen-binding enzyme. However, this substitution impeded the association of NO and destabilized the NO.heme complex. The tyrosine in the distal heme pocket also impeded both the binding and dissociation of the CO ligand. We propose that the mechanism of oxygen exclusion by sGC not only involves the lack of hydrogen bonding in the distal heme pocket, but also depends on structural elements from other domains of sGC.     

Thermotolerance and gene expression following heat stress in the whitefly Bemisia tabaci B and Q biotypes

The whitefly Bemisia tabaci (Gennadius) causes tremendous losses to agriculture by direct feeding on plants and by vectoring several families of plant viruses. The B. tabaci species complex comprises over 10 genetic groups (biotypes) that are well defined by DNA markers and biological characteristics. B and Q are amongst the most dominant and damaging biotypes, differing considerably in fecundity, host range, insecticide resistance, virus vectoriality, and the symbiotic bacteria they harbor. We used a spotted B. tabaci cDNA microarray to compare the expression patterns of 6000 ESTs of B and Q biotypes under standard 25 °C regime and heat stress at 40 °C. Overall, the number of genes affected by increasing temperature in the two biotypes was similar. Gene expression under 25 °C normal rearing temperature showed clear differences between the two biotypes: B exhibited higher expression of mitochondrial genes, and lower cytoskeleton, heat-shock and stress-related genes, compared to Q. Exposing B biotype whiteflies to heat stress was accompanied by rapid alteration of gene expression. For the first time, the results here present differences in gene expression between very closely related and sympatric B. tabaci biotypes, and suggest that these clear-cut differences are due to better adaptation of one biotype over another and might eventually lead to changes in the local and global distribution of both biotypes.     

Intraocular pressure profile during the modified diurnal tension curve using Goldman applanation tonometry and dynamic contour tonometry

The aim of this study was to compare the intraocular pressure (IOP) profile during the modified diurnal tension curve (mDTC) using Goldman applanation tonometry (GAT) and dynamic contour tonometry (DCT) in treated glaucomatous eyes. Eligible subjects were submitted to the mDTC using GAT and DCT in this sequence. IOP measurements were performed at 8 a.m., 10 a.m., 2 p.m., and 4 p.m.. Central corneal thickness was measured using ultrasound pachymetry in the morning. Statistical analysis was performed using paired Student’s t test and Bland–Altman plot. The mean difference between DCT and GAT measurements was 0.9 mmHg. The mean ± SD IOP measurements during the mDTC were 19.68 ± 4.68, 17.63 ± 4.44, 17.25 ± 5.41, and 17.32 ± 4.25 mmHg using GAT and 19.97 ± 4.75, 18.79 ± 4.61, 19.53 ± 5.30, and 19.43 ± 5.45 mmHg using DCT. IOP measurements were higher in the morning (8 a.m.) and decreased throughout the day using both tonometers. The difference between IOP measurements using GAT and DCT was smaller in the morning and increased throughout the day. The IOP variability using GAT was higher than using DCT. Corneal biomechanical properties might help explain our findings.     

The placental cholinergic system: localization to the cytotrophoblast and modulation of nitric oxide

Background

The human placenta, a non-neuronal tissue, contains an active cholinergic system comprised of acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and high affinity muscarinic receptors. The cell(s) of origin of placental ACh and its role in trophoblast function has not been defined. These studies were performed to define the cellular location of ACh synthesis (ChAT) in the human placenta and to begin studying its functional role.

Results

Using immunohistochemical techniques, ChAT was observed primarily within the cytotrophoblasts of preterm placentae as well as some mesenchymal elements. Similar intense immunostaining of the cytotrophoblast was observed for endothelium-derived nitric oxide synthase (eNOS) suggesting that ACh may interact with nitric oxide (NO)-dependent signaling pathways. The ability of carbamylcholine (CCh), an ACh analogue, to stimulate a rise in intracellular Ca⁺⁺ and NO production in trophoblasts was therefore tested using the BeWo^b30 choriocarcinoma cell as a model system. First, CCh significantly increased intracellular calcium as assessed by fluorescence microscopy. We then examined the ability of CCh to stimulate NO production by measuring total nitrite/nitrate production in conditioned media using chemiluminescence-based analysis. CCh, alone, had no effect on NO production. However, CCh increased measurable NO approximately 100% in the presence of 10 nM estradiol. This stimulatory effect was inhibited by 1 (micro)M scopolamine suggesting mediation via muscarinic receptors. Estradiol, alone, had no effect on total NO or eNOS protein or mRNA.

Conclusion

These data demonstrate that placental ChAT localizes to the cytotrophoblast and some mesenchymal cells in human placenta. It further suggests that ACh acts via muscarinic receptors on the trophoblast cell membrane to modulate NO in an estrogen-dependent manner.     

Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells

Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 approximately 100 microM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.     

Quantitative protein profiling in heart mitochondria from diabetic rats

Quantitative protein profiling based on in vitro stable isotope labeling, two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry is an accurate and reliable approach to measure simultaneously the relative abundance of many individual proteins within two different samples. In the present study, it was used to define a set of alterations caused by diabetes in heart mitochondria from streptozotocin-treated rats. We demonstrated that the expression of proteins from the myocardial tricarboxylic acid cycle was not altered in diabetes. However, up-regulation of the fatty acid beta-oxidation favored fatty acids over glucose as a source of acetyl CoA for the tricarboxylic acid cycle. Protein levels for several proteins involved in electron transport were modestly decreased. Whether this may depress overall ATP production remains to be established, since the protein level of ATP synthase seems to be unchanged. Other changes include down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75. The mitochondria-associated level of albumin was decreased, while the level of catalase was substantially increased. All of the changes were evident as early as 1 week after streptozotocin administration. Taken together, these data point to a rapid and highly coordinated regulation of mitochondrial protein expression that occurs during the heart adaptation to diabetes.     

Honokiol,a small molecular weight natural product,inhibits angiogenesis in vitro and tumor growth in vivo

Natural products comprise a major source of small molecular weight angiogenesis inhibitors. We have used the transformed endothelial cell line SVR as an effective screen of natural product extracts to isolate anti-angiogenesis and anti-tumor compounds. Aqueous extracts of Magnolia grandiflora exhibit potent activity in our SVR proliferation assays. We found that the small molecular weight compound honokiol is the active principle of magnolia extract. Honokiol exhibited potent anti-proliferative activity against SVR cells in vitro. In addition, honokiol demonstrated preferential inhibition of primary human endothelial cells compared with fibroblasts and this inhibition was antagonized by antibodies against TNF alpha-related apoptosis-inducing ligand. In vivo, honokiol was highly effective against angiosarcoma in nude mice. Our preclinical data suggests that honokiol is a systemically available and non-toxic inhibitor of angiogenesis and should be further evaluated as a potential chemotherapeutic agent.