Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice

TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo.     

Structural insights into the anti-cancer activity of quercetin on G-tetrad,mixed G-tetrad,and G-quadruplex DNA using quantum chemical and molecular dynamics simulations

Abstract

Human telomerase referred as ‘terminal transferase’ is a nucleoprotein enzyme which inhibits the disintegration of telomere length and act as a drug target for the anticancer therapy. The tandem repeating structure of telomere sequence forms the guanine-rich quadruplex structures that stabilize stacked tetrads. In our present work, we have investigated the interaction of quercetin with DNA tetrads using DFT. Geometrical analysis revealed that the influence of quercetin drug induces the structural changes into the DNA tetrads. Among DNA tetrads, the quercetin stacked with GCGC tetrad has the highest interaction energy of ?88.08?kcal/mol. The binding mode and the structural stability are verified by the absorption spectroscopy method. The longer wavelength was found at 380?nm and it exhibits bathochromic shift. The findings help us to understand the binding nature of quercetin drug with DNA tetrads and it also inhibits the telomerase activity. Further, the quercetin drug interacted with G-quadruplex DNA by using molecular dynamics (MD) simulation studies for 100?ns simulation at different temperatures and different pH levels (T?=?298 K, 320?K and pH = 7.4, 5.4). The structural stability of the quercetin with G-quadruplex structure is confirmed by RMSD. For the acidic condition (pH = 5.4), the binding affinity is higher toward G-quadruplex DNA, this result resembles that the quercetin drug is well interacted with G-quadruplex DNA at acidic condition (pH = 7.4) than the neutral condition. The obtained results show that quercetin drug stabilizes the G-quadruplex DNA, which regulates telomerase enzyme and it potentially acts as a novel anti-cancer agent.

Communicated by Ramaswamy H. Sarma     

Molecular docking analysis of phytocompounds from Andrographis paniculata binding with proteins in the notch-signaling pathway

It is of interest to document the molecular docking analysis of phytocompounds from Andrographis paniculata binding with protein NOTCH1 in the Notch-signaling pathway in the context of cancer. Hence, we document the binding features of neoandrographolide, 14-deoxyandrographolide, androgapholide and andrograpanin with proteins in the notch-signaling pathway for further consideration.     

Bioluminescence Imaging Reveals Dynamics of Beta Cell Loss in the Non-Obese Diabetic (NOD) Mouse Model

We generated a mouse model (MIP-Luc-VU-NOD) that enables non-invasive bioluminescence imaging (BLI) of beta cell loss during the progression of autoimmune diabetes and determined the relationship between BLI and disease progression. MIP-Luc-VU-NOD mice displayed insulitis and a decline in bioluminescence with age which correlated with beta cell mass, plasma insulin, and pancreatic insulin content. Bioluminescence declined gradually in female MIP-Luc-VU-NOD mice, reaching less than 50% of the initial BLI at 10 weeks of age, whereas hyperglycemia did not ensue until mice were at least 16 weeks old. Mice that did not become diabetic maintained insulin secretion and had less of a decline in bioluminescence than mice that became diabetic. Bioluminescence measurements predicted a decline in beta cell mass prior to the onset of hyperglycemia and tracked beta cell loss. This model should be useful for investigating the fundamental processes underlying autoimmune diabetes and developing new therapies targeting beta cell protection and regeneration.     

Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.     

The effects of hitchhiker antigens co-eluting with affinity-purified research antibodies

The popularity of Protein G for the purification of antibodies has given rise to an entire industry that supplies scientists with research grade immunoreagents; however, many times the supplied product is contaminated with antigens bound to the antibody's complementarity-determining regions (CDRs). These "hitchhikers" are a category of host cell proteins that are elusive to detect due to their interaction with the antibody in the final product and yet their impact on an experiment or an entire field of study can be far reaching. In an earlier work, the role of hitchhikers on a human anti-histone antibody destined for clinical usage was explored and a stringent purification scheme developed. Here we use a murine monoclonal, which reflects the type of commercial antibody usually purchased for research. We evaluate three purification schemes: a traditional approach using a one-step, low pH elution buffer (pH 2.5); a gentler approach using a pH gradient elution scheme (pH 7 down to pH 2.5); and finally, a more stringent purification patterned on our earlier published method that uses a quaternary amine guard column and a high salt wash during antibody immobilization on the Protein G. We stress that the stringent purification incorporates the pH gradient scheme and is gentler than the low-pH approach. The resulting product from all three purifications is directly compared for binding potency, histone content (using an ELISA based assay) and residual DNA (using quantitative PCR). The results demonstrate that the first two methods are inadequate for hitchhiker removal. The traditional one-step, low pH approach produces a single elution peak containing histone contaminated antibody with picogram quantities of residual DNA, however, the trailing end of the same peak is loaded with antibody complexed to nanogram amounts of DNA, in some cases, over 100 ng. The pH gradient approach provided antibodies accompanied by only picograms of residual DNA and, on average, 1 out of every 10-20 CDRs occupied by a histone antigen. The more stringent approach, using the salt wash prior to elution with the pH gradient, has an average of 1 out of every 75 CDRs contaminated with a histone while the majority of the residual DNA is captured by the quaternary amine column placed in front of the Protein G. The consequences of these contaminants is illustrated by showing how they manifest themselves in unusual antibody potency values ranging from 558% for antibody bound to histone hitchhikers down to 15% for antibody contaminated with DNA hitchhikers. Those samples purified by the recommended stringent approach show potency values between 90 and 101%. Most importantly, we repeatedly demonstrate in a simulated chromatin immunoprecipitation (ChIP) assay the ability to precipitate clean plasmid DNA with histone contaminated antibody that had been purified using the traditional one-step, low pH elution approach. Expectedly, those antibodies stringently purified and showing 100% binding potency were unable to precipitate DNA in the absence of histone hitchhikers.     

Insulin Receptor Signaling in Cones

In humans, age-related macular degeneration and diabetic retinopathy are the most common disorders affecting cones. In retinitis pigmentosa (RP), cone cell death precedes rod cell death. Systemic administration of insulin delays the death of cones in RP mouse models lacking rods. To date there are no studies on the insulin receptor signaling in cones; however, mRNA levels of IR signaling proteins are significantly higher in cone-dominant neural retina leucine zipper (Nrl) knock-out mouse retinas compared with wild type rod-dominant retinas. We previously reported that conditional deletion of the p85α subunit of phosphoinositide 3-kinase (PI3K) in cones resulted in age-related cone degeneration, and the phenotype was not rescued by healthy rods, raising the question of why cones are not protected by the rod-derived cone survival factors. Interestingly, systemic administration of insulin has been shown to delay the death of cones in mouse models of RP lacking rods. These observations led to the hypothesis that cones may have their own endogenous neuroprotective pathway, or rod-derived cone survival factors may be signaled through cone PI3K. To test this hypothesis we generated p85α^−/−/Nrl^−/− double knock-out mice and also rhodopsin mutant mice lacking p85α and examined the effect of the p85α subunit of PI3K on cone survival. We found that the rate of cone degeneration is significantly faster in both of these models compared with respective mice with competent p85α. These studies suggest that cones may have their own endogenous PI3K-mediated neuroprotective pathway in addition to the cone viability survival signals derived from rods.     

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.     

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.