Juxtaposition of the steroid binding domain-like I and II regions constitutes a ligand binding site in the sigma-1 receptor

sigma-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542 ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933 ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[125I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig sigma-1 receptor (Cys94) and at position 190 in the SBDLII region of a mutant guinea pig sigma-1 receptor (C94A,V190C), both in a sigma-ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the sigma-1 receptor.     

Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis

Rv3868, a conserved hypothetical protein of the ESAT-6 secretion system of Mycobacterium tuberculosis, is essential for the secretion of at least four virulence factors. Each protein chain is approximately 63 kDa and assembles into a hexamer. Limited proteolysis demonstrates that it consists of two domains joined by a linker. The N-terminal domain is a compact, helical domain of approximately 30 kDa and apparently functions to regulate the ATPase activity of the C-terminal domain and the oligomerization. The nucleotide binding site is situated in the C-terminal domain, which exhibits ATP-dependent self-association. It is also the oligomerization domain. Dynamic fluorescence quenching studies demonstrate that the domain is proximal to the C terminus in the apoprotein and exhibits a specific movement upon ATP binding. In silico modeling of the domains suggests that Arg-429 of a neighboring subunit forms a part of the binding site upon oligomerization. Mutational analysis of binding site residues demonstrates that the Arg-429 functions as the important "sensor arginine" in AAA-ATPases. Protein NMR experiments involving CFP-10 and activity assays rule out a general chaperone-like function for Rv3868. On the other hand, ATP-dependent "open-close" movements of the individual domains apparently enable it to interact and transfer energy to co-proteins in the ESX-1 pathway.     

Effect of inorganic N on the population,in vitro colonization and morphology of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus)

We investigated whether Acetobacter diazotrophicus (syn.Gluconacetobacter diazotrophicus) could be recovered only from sugarcane plants either with low or no application of fertiliser N. We report here the enrichment and enumeration of A. diazotrophicus from high N-fertilised samples where high heterotrophic populations reduce the numbers of A. diazotrophicus ultimately diminshing its isolation frequency as reported earlier. The growth medium of micropropagated sugarcane seedlings of the varieties Co 8021, Co 86249, Co 86010, Co 86032, and Co 87025 was amended with potassium nitrate, ammonium nitrate, ammonium chloride and urea. The colonisation and AR activity of A. diazotrophicus were affected in the presence of high levels (25 mM) of ammonium chloride and ammonium nitrate but remained unaffected in low levels of N (i.e 1/10th of MS liquid medium) and with high levels of potassium nitrate (25 mM) and urea (500 ppm). A. diazotrophicus was detected in the inoculated plants both at low and high levels of N based on the amplification of a specific 16S rRNA gene fragment using PCR based method targeting a stretch of 445 bp with primers AC and DI. High levels of N in the growth medium induced morphological changes on A. diazotrophicus cells resulting in long pleomorphic cells. The percentage of pleomorphic cells was in the decending order from NH₄NO₃, NH₄Cl, KNO₃, and urea. These changes were more prominent in ammonium chloride and ammonium nitrate than potassium nitrate, urea and N free medium. The morphological changes and the increased heterotrophic populations may play a role on the survival ofA. diazotrophicus in high N-fertilised samples/environments.     

An insight into anti-diabetic properties of dietary phytochemicals

Diabetes are a serious metabolic diseases characterized by abnormally increased plasma glucose levels. Botanical food products have been used for combating human diseases for thousands of years, since they exhibit a wide range of biological properties that can be exploited for medical application. Phytochemicals from foods, including flavonoids, monoterpenes, terpenoids, stilbenes, lignans, coumarins, alkaloids, and others, have been proposed as effective supplements for diabetes management and prevention of its long-term complications in vitro and in vivo. The mechanism of antidiabetic activity of dietary phytochemicals could be summarized as: stimulation of insulin secretion from pancreatic β-cell, reduction of insulin resistance and increasing of insulin sensitivity, stimulation of glycogenesis and hepatic glycolysis, activation of PPARγ, anti-inflammatory and antioxidant effects, inhibition of α-amylase, β-galactocidase and α-glucocidase, and inhibition intestinal absorption of glucose. Therefore the anti-diabetic effects and their mechanism of action of phytochemicals from botanical foods used traditionally in the treatment of diabetes mellitus are critically reviewed.     

Studies on Core-Shell Nanocapsules of Felodipine: <Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluations

The present study aimed for in vitro-in vivo-in silico simulation studies of experimentally designed (3²-factorial) Capmul PG-8-cored, Eudragit RSPO-Lutrol F 127 nanocapsules to ferry felodipine using GastroPlus?. The in silico parameter sensitivity analysis for pharmacokinetic parameters was initially assessed to justify the preparation of felodipine-loaded nanocapsules (FLNs) with enhanced solubility to overcome the bioavailability issues of felodipine. The overall integrated desirability ranged between 0.8187 and 0.9488 for three optimized FLNs when analyzed for mean particle size, zeta potential, encapsulation efficiency, and in vitro dissolution parameters. The morphological evaluation (SEM, TEM, and AFM) demonstrated spherical nanoparticles (200–300 nm). Validated LC-MS/MS analysis demonstrated enhanced relative bioavailability (13.37-fold) of optimized FLN as compared to suspension. The simulated regional absorption of the FLN presented significant absorption from the cecum (26.3%) and ascending colon (20.1%) with overall absorption of 67.4% from the GIT tract. Furthermore, in vitro-in vivo correlation demonstrated the Wagner-Nelson method as the preferred model as compared to mechanistic and numerical deconvolution on the basis of least mean absolute prediction error, least standard error of prediction, least mean absolute error, and maximum correlation coefficient (r ² = 0.920). The study demonstrated enhanced oral absorption of felodipine-loaded nanocapsules, and GastroPlus? was found to be an efficient simulation tool for in vitro-in vivo-in silico simulations.     

HER2-mediated internalization of a targeted prodrug cytotoxic conjugate is dependent on the valency of the targeting ligand

HER2 is a validated therapeutic target for cancer. There are no natural ligands, but monoclonal antibodies and peptides that bind HER2 act as artificial ligands, selectively affecting HER2-overexpressing tumors. One reported mechanism for this effect is receptor downregulation, but the expected correlation of ligand-dependent HER2 internalization and tumor inhibition remain poorly characterized. Moreover, HER2 ligands have limited therapeutic efficacy and often they require adjuvant treatment with the chemotherapeutic Taxol. Here, we generated a series of HER2 ligands (Anti-HER2/neu peptide ligands, AHNPmonovalent and AHNPbivalent) with different valency and correlated their internalization-promoting ability to biological potency. Since AHNPbivalent (but not AHNPmonovalent) induces rapid receptor internalization, we exploited this feature to deliver cytotoxic conjugates coupling AHNPbivalent and Taxol (Taxol . AHNPbivalent). The prodrug conjugate releases Taxol after receptor-mediated internalization, and cytotoxicity can be used as a marker of internalization. Taxol . AHNPbivalent is significantly more cytotoxic than free Taxol + free AHNPbivalent. Hence, the Taxol x AHNP(bivalent) prodrug binds to HER2, induces receptor internalization and downregulation, and the subsequent release of free Taxol inside the targeted cell results in synergistic toxicity, The effect is selective towards HER2- expressing cells. This work links HER2 receptor internalization and growth arrest, and the chemical conjugation strategy may yield improved and HER2 selective therapeutics.     

Individual differences among grapheme-color synesthetes: brain-behavior correlations

Grapheme-color synesthetes experience specific colors associated with specific number or letter characters. To determine the neural locus of this condition, we compared behavioral and fMRI responses in six grapheme-color synesthetes to control subjects. In our behavioral experiments, we found that a subject's synesthetic experience can aid in texture segregation (experiment 1) and reduce the effects of crowding (experiment 2). For synesthetes, graphemes produced larger fMRI responses in color-selective area human V4 than for control subjects (experiment 3). Importantly, we found a correlation within subjects between the behavioral and fMRI results; subjects with better performance on the behavioral experiments showed larger fMRI responses in early retinotopic visual areas (V1, V2, V3, and hV4). These results suggest that grapheme-color synesthesia is the result of cross-activation between grapheme-selective and color-selective brain areas. The correlation between the behavioral and fMRI results suggests that grapheme-color synesthetes may constitute a heterogeneous group.     

Neurocognitive mechanisms of synesthesia

Synesthesia is a condition in which stimulation of one sensory modality causes unusual experiences in a second, unstimulated modality. Although long treated as a curiosity, recent research with a combination of phenomenological, behavioral, and neuroimaging methods has begun to identify the cognitive and neural basis of synesthesia. Here, we review this literature with an emphasis on grapheme-color synesthesia, in which viewing letters and numbers induces the perception of colors. We discuss both the substantial progress that has been made in the past fifteen years and some open questions. In particular, we focus on debates in the field relating to the neural basis of synesthesia, including the relationship between synesthesia and attention and the role of meaning in synesthetic colors. We propose that some, but probably not all, of these differences can be accounted for by differences in the synesthetes studied and discuss some methodological implications of these individual differences.     

Estrogenic regulation of tissue factor and tissue factor pathway inhibitor in platelets

Oral estrogen treatment increases thrombotic risk. Tissue factor (TF), tissue factor pathway inhibitor (TFPI), and platelet interaction with leukocytes are important determinants of thrombogenesis. Therefore, the present study was designed to define and compare platelet TF and TFPI mRNA and adhesion protein expression in platelets derived from animals treated with different types of oral estrogens. Ovariectomized pigs were treated with 17beta-estradiol (2 mg/day), conjugated equine estrogen (CEE; 0.625 mg/day), or raloxifene (60 mg/day) for 4 wk. Compared with intact animals, ovariectomy and treatment differentially affected populations of leukocytes: neutrophils decreased whereas lymphocytes increased significantly 4 wk after ovariectomy and with 17beta-estradiol and CEE treatments; eosinophils increased only with 17beta-estradiol treatment. Content of TF protein increased in platelets from 17beta-estradiol- and raloxifene-treated pigs, whereas TF mRNA was detected only in platelets from 17beta-estradiol- and CEE treated pigs. TFPI mRNA increased in platelets after ovariectomy and estrogen treatment. Only a trace of TFPI protein was detected, but a higher-molecular-mass protein was observed in all treatment groups. Expression of CD40 and CD40 ligand increased with ovariectomy and decreased with 17beta-estradiol and CEE treatments more than with raloxifene. The ratio of activated to basal P-selectin expression decreased with ovariectomy and increased with raloxifene treatments. These results suggest that estrogenic formulations may affect individual thrombotic risk by different mechanisms that regulate TF and platelet-leukocytic interactions. These studies provide the rationale for evaluation of interactions among platelets and TF and TFPI expression on thrombin generation during estrogen treatment in humans.