Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists. Effect of basal desensitization.

In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.     

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.     

Relaxin and β-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by β-estradiol. We postulated that relaxin and β-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, β-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered β-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 μg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. β-estradiol, relaxin, or β-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or β-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by β-estradiol, relaxin, or β-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

Structure-radical scavenging activity relationships of flavonoids

The objective of this work is to establish the structural requirements of flavonoids for appreciable radical-scavenging activity (RSA) and elucidate a comprehensive mechanism that can explain their activity. To this end, the RSA of 52 flavonoids against 2,2-diphenyl-1-picrylhydrazyl was determined. The relative change in energy (DeltaH(f)) associated with the formation of various flavonoidal and other phenolic radicals and also the spin distribution in these radicals were determined using computational programmes. By correlating experimental data with DeltaH(f), structural features that affect activity have been identified and considered in perspective. It was shown with compelling evidences that the RSA of flavonoids could be mapped to one of their ring systems, making it possible to study their RSA by dissecting their structures and designing representative simpler models. Consequently, hydroxytoluene units were demonstrated to successfully account for the RSA of flavonoids due to ring B and also to satisfactorily do so for activities due to ring A. Further, a comprehensive model for the radical scavenging reactions of flavonoids (and in general, phenolic compounds), which could account for hydrogen atom donation and the termination of aroxyl radicals, was proposed. Finally, prediction of structural features that could endow flavonoids with appreciable radical scavenging capability was made by considering the stability data and the ease of termination. In conclusion, the underlying molecular phenomena of the RSA of flavonoids could be explained by the ease of hydrogen atom abstraction and the ease of the termination of the flavonoidal aroxyl radicals.     

Design,synthesis, molecular docking and anti-hepatocellular carcinoma evaluation of novel acyclic pyridine thioglycosides

A novel series of acyclic pyridine thioglycosides has been synthesized. Evaluation of the anti proliferative activity of these compounds against HEPG-2 cell lines (liver carcinoma cell lines) shows that most of the compounds have high anti-tumor activities especially 6b, 6c, 7b and 7c. Furthermore, in the modeling study, these compounds showed that they have high binding affinity with thymidylate synthase dihydrofolate reductase (TS-DHFR).     

Average beta burst duration profiles provide a signature of dynamical changes between the ON and OFF medication states in Parkinson’s disease

Parkinson’s disease motor symptoms are associated with an increase in subthalamic nucleus beta band oscillatory power. However, these oscillations are phasic, and there is a growing body of evidence suggesting that beta burst duration may be of critical importance to motor symptoms. This makes insights into the dynamics of beta bursting generation valuable, in particular to refine closed-loop deep brain stimulation in Parkinson’s disease. In this study, we ask the question “Can average burst duration reveal how dynamics change between the ON and OFF medication states?”. Our analysis of local field potentials from the subthalamic nucleus demonstrates using linear surrogates that the system generating beta oscillations is more likely to act in a non-linear regime OFF medication and that the change in a non-linearity measure is correlated with motor impairment. In addition, we pinpoint the simplest dynamical changes that could be responsible for changes in the temporal patterning of beta oscillations between medication states by fitting to data biologically inspired models, and simpler beta envelope models. Finally, we show that the non-linearity can be directly extracted from average burst duration profiles under the assumption of constant noise in envelope models. This reveals that average burst duration profiles provide a window into burst dynamics, which may underlie the success of burst duration as a biomarker. In summary, we demonstrate a relationship between average burst duration profiles, dynamics of the system generating beta oscillations, and motor impairment, which puts us in a better position to understand the pathology and improve therapies such as deep brain stimulation.     

Developmental stages of Hepatozoon hemprichii sp. nov. infecting the skink Scincus hemprichii and the tick Hyalomma impeltatum from Saudi Arabia

The life cycle of Hepatozoon hemprichi n. sp. is described; the vertebrate host is Scincus hemprichii and it is vectored by Hyalomma impeltatum. Erythrocytic stages of 18 ± 1.8 × 4 ± 0.8 μm developed in the hemocoel of ticks to sporozoites within 16-18 days. Schizogony occurred in the liver parenchyma and the endothelial cells of blood capillaries in lung and spleen. Mature schizonts measuring 27 ± 3.11 × 20.13 ± 3.0 μm produced 28 merozoites (on average). The merozoites were 13 ± 1.21 × 1.21 ± 0.72 μm with nuclei 5 ± 0.65 × 2.1 ± 0.51 μm. Syzygy and differentiation of gamonts took place in tick's hemocoel up to the third day post-infection (PI). The microgamont (16 ± 0.31 × 18 ± 0.42 μm) produced 4, uniflagellated microgametes at 4-5 days PI. The microgamete measured 15.2 ± 0.31 μm while the flagellum was always at least 26 μm. The macrogamete was very large in size (31 ± 3.11 μm) with a central nucleus. After fertilization, (5-6 days PI) zygotes developed into oocysts (55 ± 3.41 × 52 ± 4.11 μm) in which repeated mitotic divisions with centripetal invaginations occurred; each contained 18 banana-shaped sporozoites, 13.61 ± 0.8 × 1.2 ± 0.31 μm in size. Experimental transmission was successfully carried out by oral administration or by intra-peritoneal inoculation of the infective stages (sporozoites) to uninfected skinks and led to the appearance of blood stages after 5 wk and 4 wk, respectively.     

Tailoring FeN4 Sites with Edge Enrichment for Boosted Oxygen Reduction Performance in Proton Exchange Membrane Fuel Cell

Transition metal atoms with corresponding nitrogen coordination are widely proposed as catalytic centers for the oxygen reduction reaction (ORR) in metal–nitrogen–carbon (M–N–C) catalysts. Here, an effective strategy that can tailor Fe–N–C catalysts to simultaneously enrich the number of active sites while boosting their intrinsic activity and utilization is reported. This is achieved by edge engineering of FeN₄ sites via a simple ammonium chloride salt‐assisted approach, where a high fraction of FeN₄ sites are preferentially generated and hosted in a graphene‐like porous scaffold. Theoretical calculations reveal that the FeN₄ moieties with adjacent pore defects are likely to be more active than the nondefective configuration. Coupled with the facilitated accessibility of active sites, this prepared catalyst, when applied in a practical H₂–air proton exchange membrane fuel cell, delivers a remarkable peak power density of 0.43 W cm^?2, ranking it as one of the most active M–N–C catalysts reported to date. This work provides a new avenue for boosting ORR activity by edge manipulation of FeN₄ sites.     

Simultaneous determination of metolazone and losartan in their combined tablets using synchronous fluorescence spectroscopy

Synchronous spectrofluorimetry is utilized to carry out a rapid, sensitive and reliable method for determination of the binary mixture of metolazone (MTL) and losartan potassium (LSP). Under optimized experimental conditions, the synchronized fluorescence spectra of the two drugs were measured at Δλ = 80 nm in acidic methanolic solution and intensities were recorded at 260 nm for MTL and 335 nm for LSP. Linear correlation between fluorescence intensity and concentration were obtained through the ranges 0.02–0.2 μg/mL and 0.2–2.0 μg/mL for MTL and LSP, respectively. Limits of detection were 3.02 and 0.12 ng/mL, whereas limits of quantification were 9.16 and 0.35 ng/mL for MTL and LSP, respectively. The designated procedure was easily and successfully adopted to determine the two compounds in their single, as well as in co‐formulated, tablets and the results showed high precision and accuracy without any significant interference from common tablet excipients. A comparison of the obtained results with a published reference method was carried out and both showed good agreement with respect to accuracy and precision.     

Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st(-/-)) by embryonic stem cell technology. C6st(-/-) mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st(-/-) mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, whereas the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. Despite the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st(-/-) mice. Further analysis revealed that the number of CD62L(+)CD44(low) T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st(-/-) mice was significantly decreased, whereas those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.