Single, 14-Day, and 13-Week Repeated Dose Toxicity Studies of Daily Oral Gelidium elegans Extract Administration to Rats.

Gelidium elegans extract (GEE) is derived from a red alga from the Asia-Pacific region, which has antioxidant, anti-adipogenic, and anti-hyperglycemic effects. However, detailed studies of the toxicology of GEE have not been performed. We evaluated the single oral dose toxicity of GEE in male and female Sprague-Dawley (CD) rats. GEE did not cause deaths or have toxic effects at dosages of 5000 mg/kg/day, although compound-colored stools and diarrhea were observed in both sexes, which lasted <2 days. Therefore, the LD50 of GEE is likely to be >5000 mg/kg. We next evaluated the repeated oral dose toxicity of GEE in CD rats over 14 days and 13 weeks. GEE did not induce any significant toxicological changes in either sex at 2000 mg/kg/day. Repeated oral dose toxicity studies showed no adverse effects, in terms of clinical signs, mortality, body mass, food consumption, ophthalmic examination, urinalysis, hematology, serum biochemistry, necropsy, organ masses, or histopathology, at dosages of 500, 1000, or 2000 mg/kg/day. The no observed adverse effect level (NOAEL) for GEE is thus likely to be >2000 mg/kg/day, and no pathology was identified in potential target organs. Therefore, this study indicates that repeated oral dosing with GEE is safe in CD rats.

Caffeine prevents LPS-induced inflammatory responses in RAW264.7 cells and zebrafish.

Caffeine is a white crystalline xanthine alkaloid found in the seeds of coffee plants and leaves of the tea bush. In this study, we evaluated whether caffeine exerts anti-inflammatory effects on lipopolysaccharide (LPS)-induced inflammation both in vitro and in vivo. RAW264.7 cells were treated with various concentrations of caffeine in the presence or absence of LPS. Caffeine decreased the LPS-induced inflammatory mediator, nitric oxide (NO). Caffeine treatment also reduced the expression of pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-3, IL-6 and IL-12, and decreased both IL-6 secretion and phosphorylated p38MAPK expression in LPS-treated RAW264.7 cells. Caffeine inhibited nuclear translocation of nuclear factor κB (NF-κB) via IκBα phosphorylation. In addition, caffeine inhibited LPS-induced NO production in zebrafish. These results suggest that caffeine may suppress LPS-induced inflammatory responses in RAW264.7 cells by regulating NF-κB activation and MAPK phosphorylation.

Stability of tacrolimus solutions in polyolefin containers.

PURPOSE: Results of a study to determine the stability of tacrolimus solutions stored in polyolefin containers under various temperature conditions are reported. METHODS: Triplicate solutions of tacrolimus (0.001, 0.01, and 0.1 mg/mL) in 0.9% sodium chloride injection or 5% dextrose injection were prepared in polyolefin containers. Some samples were stored at room temperature (20-25 °C); others were refrigerated (2-8 °C) for 20 hours and then stored at room temperature for up to 28 hours. The solutions were analyzed by stability-indicating high-performance liquid chromatography (HPLC) assay at specified time points over 48 hours. Solution pH was measured and containers were visually inspected at each time point. Stability was defined as retention of at least 90% of the initial tacrolimus concentration. RESULTS: All tested solutions retained over 90% of the initial tacrolimus concentration at all time points, with the exception of the 0.001-mg/mL solution prepared in 0.9% sodium chloride injection, which was deemed unstable beyond 24 hours. At all evaluated concentrations, mean solution pH values did not change significantly over 48 hours; no particle formation was detected. CONCLUSION: During storage in polyolefin bags at room temperature, a 0.001-mg/mL solution of tacrolimus was stable for 24 hours when prepared in 0.9% sodium chloride injection and for at least 48 hours when prepared in 5% dextrose injection. Solutions of 0.01 and 0.1 mg/mL prepared in either diluent were stable for at least 48 hours, and the 0.01-mg/mL tacrolimus solution was also found to be stable throughout a sequential temperature protocol.

Oleuropein suppresses LPS-induced inflammatory responses in RAW 264.7 cell and zebrafish.

Oleuropein is one of the primary phenolic compounds present in olive leaf. In this study, the anti-inflammatory effect of oleuropein was investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 and a zebrafish model. The inhibitory effect of oleuropein on LPS-induced NO production in macrophages was supported by the suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, our enzyme immunoassay showed that oleuropein suppressed the release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Oleuropein inhibited the translocation of p65 by suppressing phosphorylation of inhibitory kappa B-α (IκB-α). Oleuropein also decreased activation of ERK1/2 and JNK, which are associated with LPS-induced inflammation, and its downstream gene of AP-1. Furthermore, oleuropein inhibited LPS-stimulated NO generation in a zebrafish model. Taken together, our results demonstrated that oleuropein could reduce inflammatory responses by inhibiting TLR and MAPK signaling, and may be used as an anti-inflammatory agent.

Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate.

HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K(+) channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K(+) channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd(2+) ions freeze the open-closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd(2+) and measure the effect on voltage sensor movement. Cd(2+) did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd(2+)-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open-closed equilibrium, corresponding to a coupling energy of ∼1.3-2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K(+) channels, but also much weaker.

Uncoupling proton activation of vanilloid receptor TRPV1.

Multimodal gating is an essential feature of many TRP ion channels, enabling them to respond to complex cellular environments. TRPV1, a pain receptor involved in nociception at the peripheral nerve terminals, can be activated by a range of physical and chemical stimuli (e.g., capsaicin, proton, and heat) and further sensitized by proinflammatory substances. How a single receptor achieves this multiplicity of functionality is poorly understood at the molecular level. Here, we investigated the structural basis of proton activation of TRPV1. Chimeric channels between rTRPV1 and the low pH-insensitive homolog TRPV2 were constructed by systematically exchanging the extracellular domains and were characterized using whole-cell recording in transiently transfected HEK293 cells. Two discrete domains, one involving the pore helix and the other the S3-S4 linker, were found crucial for direct activation of the channel by low pH. Single residue mutations in either domain (T633A/V538L) abrogated the proton-evoked current while preserving the capsaicin and heat responses and their potentiation by mildly acidic pH. Both residues exert a gating effect through hydrophobic interactions. Our results unravel novel information on the structural basis of channel function, and support the existence of discrete domains for multimodal gating of the channel. In view of the resemblance of the pore of TRPV1 to KcsA, our findings also provide evidence on the pore helix as an active component in channel gating in addition to its role in ion permeation.

Inhibitory modulation of distal C-terminal on protein kinase C-dependent phospho-regulation of rat TRPV1 receptors.

The vanilloid receptor TRPV1, previously known as VR1, has been implicated in pain sensation under both physiological and pathological conditions. The channel is highly expressed in sensory ganglion neurones and is activated by a range of noxious stimuli including irritant chemicals, acids and heat. In order to understand the structural basis underlying this polymodal activation and the regulation by intracellular signalling pathways, we have investigated the functional roles of the cytoplasmic C-terminal of rat TRPV1. A mutant with the maximal truncation of the distal C-terminal encompassing the last 88 residues was constructed. Of interest, this mutant exhibited a Ca(2+)-dependent functional loss; it was irresponsive to capsaicin in the presence of extracellular Ca(2+), but fully functional otherwise. Further studies of this construct revealed that extracellular Ca(2+) alone could activate the channel, and that the activation required protein kinase C (PKC) phosphorylation at S502, an event that was up-regulated by external Ca(2+) entry. We compared the truncation mutant with wild-type TRPV1 and demonstrated that it had a significantly increased sensitivity to PKC phosphorylation. These results suggest the distal C-terminal of TRPV1 can inhibit phosphorylation-induced potentiation of the wild-type channel. They also call into question some established functions of the distal C-terminal of TRPV1, including its roles in agonist binding and functional desensitization. We suggest that the functional loss of the truncation mutant, in the presence of extracellular Ca(2+), was not due to disruption of agonist binding or gating, but rather to desensitization promoted by unstimulated extracellular Ca(2+) entry.

Low pH potentiates both capsaicin binding and channel gating of VR1 receptors.

Capsaicin ion channels are highly expressed in peripheral nervous terminals and involved in pain and thermal sensations. One characteristic of the cloned VR1 receptor is its multimodal responses to various types of noxious stimuli. The channel is independently activated by capsaicin and related vanilloids at submicromolar range, by heat above 40 degrees C, and by protons at pH below 6.5. Furthermore, simultaneous applications of two or more stimuli lead to cross sensitization of the receptor, with an apparent increase in the sensitivity to any individual stimulus when applied alone. We studied here the mechanism underlying such cross-sensitization; in particular, between capsaicin and pH, two prototypical stimuli for the channel. By analyzing single-channel currents recorded from excised-patches expressing single recombinant VR1 receptors, we examined the effect of pH on burst properties of capsaicin activation at low concentrations and the effect on gating kinetics at high concentrations. Our results indicate that pH has dual effects on both capsaicin binding and channel gating. Lowering pH enhances the apparent binding affinity of capsaicin, promotes the occurrences of long openings and short closures, and stabilizes at least one of the open conformations of the channel. Our data also demonstrate that capsaicin binding and protonation of the receptor interact allosterically, where the effect of one can be offset by the effect of the other. These results provide important basis to further understand the nature of the activation pathways of the channel evoked by different stimuli as well as the general mechanism underling the cross-sensitization of pain.