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.     

The DPY-30 Domain and Its Flanking Sequence Mediate the Assembly and Modulation of Flagellar Radial Spoke Complexes

RIIa is known as the dimerization and docking (D/D) domain of the cyclic AMP (cAMP)-dependent protein kinase. However, numerous molecules, including radial spoke protein 2 (RSP2) in Chlamydomonas flagella, also contain an RIIa or a similar DPY-30 domain. To elucidate new roles of D/D domain-containing proteins, we investigated a panel of RSP2 mutants. An RSP2 mutant had paralyzed flagella defective in RSP2 and multiple subunits near the spokehead. New transgenic strains lacking only the DPY-30 domain in RSP2 were also paralyzed. In contrast, motility was restored in strains that lacked only RSP2's calmodulin-binding C-terminal region. These cells swam normally in dim light but could not maintain typical swimming trajectories under bright illumination. In both deletion transgenic strains, the subunits near the spokehead were restored, but their firm attachment to the spokestalk required the DPY-30 domain. We postulate that the DPY-30-helix dimer is a conserved two-prong linker, required for normal motility, organizing duplicated subunits in the radial spoke stalk and formation of a symmetrical spokehead. Further, the dispensable calmodulin-binding region appears to fine-tune the spokehead for regulation of "steering" motility in the green algae. Thus, in general, D/D domains may function to localize molecular modules for both the assembly and modulation of macromolecular complexes.     

Expression of receptors for VEGFs on normal human thyroid follicular cells and their role in follicle formation

Human thyroid follicular cells in culture expressed the mRNAs for the receptors for vascular endothelial growth factors (VEGFRs). The relative expression was neuropilin1 = neuropilin2 = VEGFR2 > VEGFR1 > VEGFR3. Western blotting for VEGFR2 showed labeling of proteins ~200-230 kDa. Clonal follicular thyroid cell lines (FRTL5 and FTC133) also expressed mRNAs for the VEGFR1 and 2 obviating concerns of endothelial cell contamination. In the primary cultures, TSH, which is essential for expression of differentiated function, reduced VEGFR2 mRNA levels by 60%. Immunostaining for VEGFRs and neuropilin2 (NRP2), showed expression on the plasma membrane but with the exception of neuropilin1 (NRP1), all VEGFRs were also found in the cytoplasm and nucleus. Antibody specific for phosphotyrosine 1214 in VEGFR2 showed that the receptor was phosphorylated in the primary cultures and the cell lines. When VEGFR signaling was blocked with a specific inhibitor, follicle formation in the primary cultures was enhanced suggesting that VEGFR activation was detrimental to follicle formation. Immunostaining of sections of normal thyroids and various pathologies showed staining for VEGFR2 and pVEGFR2. We conclude that normal thyroid follicular cell express VEGFRs. For VEGFR2 its subcellular localization suggests functions additional to that of a cell surface receptor and a role in follicular integrity.     

Intermittent Moderate Energy Restriction Improves Weight Loss Efficiency in Diet-Induced Obese Mice

Background

Intermittent severe energy restriction is popular for weight management. To investigate whether intermittent moderate energy restriction may improve this approach by enhancing weight loss efficiency, we conducted a study in mice, where energy intake can be controlled.

Methods

Male C57/Bl6 mice that had been rendered obese by an ad libitum diet high in fat and sugar for 22 weeks were then fed one of two energy-restricted normal chow diets for a 12-week weight loss phase. The continuous diet (CD) provided 82% of the energy intake of age-matched ad libitum chow-fed controls. The intermittent diet (ID) provided cycles of 82% of control intake for 5–6 consecutive days, and ad libitum intake for 1–3 days. Weight loss efficiency during this phase was calculated as (total weight change) ÷ [(total energy intake of mice on CD or ID)–(total average energy intake of controls)]. Subsets of mice then underwent a 3-week weight regain phase involving ad libitum re-feeding.

Results

Mice on the ID showed transient hyperphagia relative to controls during each 1–3-day ad libitum feeding period, and overall ate significantly more than CD mice (91.1±1.0 versus 82.2±0.5% of control intake respectively, n = 10, P<0.05). There were no significant differences between CD and ID groups at the end of the weight loss or weight regain phases with respect to body weight, fat mass, circulating glucose or insulin concentrations, or the insulin resistance index. Weight loss efficiency was significantly greater with ID than with CD (0.042±0.007 versus 0.018±0.001 g/kJ, n = 10, P<0.01). Mice on the CD exhibited significantly greater hypothalamic mRNA expression of proopiomelanocortin (POMC) relative to ID and control mice, with no differences in neuropeptide Y or agouti-related peptide mRNA expression between energy-restricted groups.

Conclusion

Intermittent moderate energy restriction may offer an advantage over continuous moderate energy restriction, because it induces significantly greater weight loss relative to energy deficit in mice.     

Wnt/β‐catenin signaling in the mouse embryonic cranial mesenchyme is required to sustain the emerging differentiated meningeal layers

Cranial neural crest cells (CNCCs) give rise to cranial mesenchyme (CM) that differentiates into the forebrain meningeal progenitors in the basolateral and apical regions of the head. This occurs in close proximity to the other CNCC‐CM‐derivatives, such as calvarial bone and dermal progenitors. We found active Wnt signaling transduction in the forebrain meningeal progenitors in basolateral and apical populations and in the non‐meningeal CM preceding meningeal differentiation. Here, we dissect the source of Wnt ligand secretion and requirement of Wnt/β‐catenin signaling for the lineage selection and early differentiation of the forebrain meninges. We find persistent canonical Wnt/β‐catenin signal transduction in the meningeal progenitors in the absence of Wnt ligand secretion in the CM or surface ectoderm, suggesting additional sources of Wnts. Conditional mutants for Wntless and β‐catenin in the CM showed that Wnt ligand secretion and Wnt/β‐catenin signaling were dispensable for specification and proliferation of early meningeal progenitors. In the absence of β‐catenin in the CM, we found diminished laminin matrix and meningeal hypoplasia, indicating a structural and trophic role of mesenchymal β‐catenin signaling. This study shows that β‐catenin signaling is required in the CM for maintenance and organization of the differentiated meningeal layers in the basolateral and apical populations of embryonic meninges.     

Lactic acid bacteria isolated from fermented flour of finger millet,its probiotic attributes and bioactive properties

This study aims to isolate and identify lactic acid bacteria from fermented flour of selected finger millet varieties grown in Sri Lanka and to evaluate their probiotic attributes and bioactive properties in vitro. Fifteen lactic acid bacteria were isolated from three varieties of fermented finger millet flour namely ravi, raavana and oshadha. These isolates were screened for phenotypical and biochemical characteristics. The selected isolates were identified by 16 S rRNA sequencing as Bacillus cereus (five strains), Streptococcus lutetiensis, Lactobacillus plantarum, Lactobacillus fermentum (two strains), Brevibacillus borstelensis, Paenibacillus species, Lactococcus lactis subspecies lactis, Enterococcus faecium, Pediococcus acidilactici, and Enterococcus lactis, and their partial sequences were deposited in GenBank. Among them, five isolates including two isolates, L. plantarum MF405176.1 and L. fermentum MF033346.1 isolated from ravi; two isolates, L. lactis MF480428.1 and E. faecium MF480431.1 isolated from raavana; and P. acidilactici MF480434.1 isolated from oshadha varieties respectively, exhibited in vitro safety attributes and could tolerate acid, gastric juice, bile, salt, phenol, and temperature under simulated gastric conditions, and also were susceptible to antibiotics tested. Further, they demonstrated bactericidal activity against both drug-sensitive and multidrug-resistant pathogens. Among the selected isolates, L. plantarum MF405176.1 demonstrated highest hydrophobicity and adhesion to both colon colorectal adenocarcinoma and colon colorectal carcinoma cell lines. L. lactis subspecies lactis MF480428.1 exhibited the highest auto-aggregation and 2, 2, diphenyl-1-pricrylhydrazyl free radical scavenging activity. P. acidilactici MF480434.1 demonstrated the lowest IC50 values against HCT-116 and HT-29 cells. None of the LAB isolates could assimilate > 10% cholesterol in vitro.     

Discovery of N-substituted pyridinones as potent and selective inhibitors of p38 kinase

The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38α. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.     

Obesity Attenuates the Contribution of African Admixture to the Insulin Secretory Profile in Peripubertal Children: A Longitudinal Analysis

The pubertal transition has been identified as a time of risk for development of type 2 diabetes, particularly among vulnerable groups, such as African Americans (AAs). Documented ethnic differences in insulin secretory dynamics may predispose overweight AA adolescents to risk for type 2 diabetes. The objectives of this longitudinal study were to quantify insulin secretion and clearance in a cohort of 90 AA and European American (EA) children over the pubertal transition and to explore the association of genetic factors and adiposity with repeated measures of insulin secretion and clearance during this critical period. Insulin sensitivity was determined by intravenous glucose tolerance test (IVGTT) and minimal modeling; insulin secretion and clearance by C‐peptide modeling; genetic ancestry by admixture analysis. Mixed‐model longitudinal analysis indicated that African genetic admixture (AfADM) was independently and positively associated with first‐phase insulin secretion within the entire group (P < 0.001), and among lean children (P < 0.01). When examined within pubertal stage, this relationship became significant at Tanner stage 3. Total body fat was a significant determinant of first‐phase insulin secretion overall and among obese children (P < 0.001). Total body fat, but not AfADM, was associated with insulin clearance (P < 0.001). In conclusion, genetic factors, as reflected in AfADM, may explain greater first‐phase insulin secretion among peripubertal AA vs. EA; however, the influence of genetic factors is superseded by adiposity. The pubertal transition may affect the development of the β‐cell response to glucose in a manner that differs with ethnic/genetic background.     

Microbial Reactions on 7α‐Hydroxyfrullanolide and Evaluation of Biotransformed Products for Antibacterial Activity

7α‐Hydroxyfrullanolide ( 1 ), a known sesquiterpenoid, was isolated from Sphaeranthus indicus using an antibacterial‐activity‐directed fractionation method. This compound had exhibited a significant antibacterial activity against Gram‐positive bacteria. Chemical and microbial reactions were performed to prepare eight different analogues of compound 1 in order to evaluate these newly synthesized compounds for antibacterial activity. These compounds were 1β,7α‐dihydroxyfrullanolide ( 2 ), 7α‐hydroxy‐1‐oxofrullanolide ( 3 ), 4,5‐dihydro‐7α‐hydroxyfrullanolide ( 4 ), 11,13‐dihydro‐7α‐hydroxyfrullanolide ( 5 ), 13‐acetyl‐7α‐hydroxyfrullanolide ( 6 ), 2α,7α‐dihydroxysphaerantholide ( 7 ), 4α,5α‐epoxy‐7α‐hydroxyfrullanolide ( 8 ), and 4β,5β‐epoxy‐7α‐hydroxyfrullanolide ( 9 ). Microbial reactions on 1 using whole‐cell cultures of Cunninghamella echinulata and Curvularia lunata yielded compounds 2 – 4 . Incubation of compound 1 with the liquid cultures of Apsergillus niger and Rhizopus circinans yielded metabolites 5 – 7 , while 8 and 9 were prepared by carrying out an epoxidation reaction on 1 using meta‐chloroperbenzoic acid (mCPBA). Structures of compounds 2 – 9 were elucidated with the aid of extensive NMR spectral studies. Compounds 2 – 4 were found to be new metabolites. Compounds 1 – 9 were evaluated for antibacterial activity and found to exhibit a wide range of bioactivities. Antibacterial‐activity data of 1 – 9 suggested that the bioactivity of 1 is largely due to the presence of C(4)?C(5), C(11)?C(13), and a γ‐lactone moiety.