Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site[s] for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB [1 mM], camphor [10 mM] and dihydrocarveol [10 mM] either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered [16 +/- 2% reduction] phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels

Human umbilical artery smooth muscle exhibits a 2-APB-sensitive capacitative contractile response evoked by vasoactive substances and expresses mRNAs for STIM, Orai and TRPC channels

After depletion of intracellular Ca2+ stores the capacitative response triggers an extracellular Ca2+ influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotonin- and histamine-induced contractions. Intracellular Ca2+ depletion by a Ca2+-free extracellular solution followed by Ca2+ readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca2+ entry. 2-APB inhibition of sarcoplasmic reticulum Ca2+ release was excluded because this blocker did not affect serotonin force development in a Ca2+-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.

Tratamiento del cáncer por captura neutrónica de boro: su aplicación al carcinoma indiferenciado de tiroides / Boron neutron capture therapy applied to undifferentiated thyroid carcinoma

El cáncer indiferenciado de tiroides es un tumor muy agresivo, de muy mal pronóstico y sin tratamiento efectivo. La terapia por captura neutrónica de boro (BNCT) podría ser una alternativa para el tratamiento de esta enfermedad. Se basa en la captación selectiva de boro por el tumor y su activación por un haz de neutrones. El boro activado libera un núcleo de litio-7 y una partícula alfa, las cuales tienen una alta transmisión linear de energía (linear energy transfer, LET) y un alcance de 5-9 µm, destruyendo el tumor. En estudios previos hemos mostrado que la línea celular humana de cáncer indiferenciado de tiroides (ARO) tiene una captación selectiva de borofenilalanina (10BPA) tanto in vitro como después de ser implantada en ratones NIH nude. También demostramos en estos animales inyectados con BPA e irradiados con un haz de neutrones térmicos, un 100% de control sobre el crecimiento tumoral y un 50% de cura histológica. En trabajos posteriores mostramos que la porfirina 10BOPP tetrakis-carborane carboxylate ester de 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin IX) cuando es inyectada 5-7 días antes que el BPA se obtiene una concentración tumoral de boro de aproximadamente el doble que el BPA solo (45-38 ppm vs. 20 ppm). La posterior irradiación con neutrones mostró un 100% de remisión completa en animales con tumores cuyo volumen pre-tratamiento era de 50 mm3 o menor. Los perros padecen CIT espontáneo, con un comportamiento biológico similar al humano, y una captación selectiva de BPA, abriendo la posibilidad de su tratamiento por BNCT

Undifferentiated thyroid carcinoma (UTC) is an aggressive tumor with a poor prognosis due to the lack of an effective treatment. Boron neutron capture therapy (BNCT) is based on the selective uptake of boron by the tumor and its activation by a neutron beam, releasing lithium-7 and an alpha particle that will kill the tumor cells by their high linear energy transfer (LET). In previous studies we have shown a selective uptake of borophenylalanine (10BPA) in a human UTC cell line (ARO) and in NIH nude mice implanted with this cell line. When these animals were injected with BPA and irradiated with an appropriated neutron beam, we observed a 100% of tumor growth control and a 50 % of histological cure when the initial tumor volume was 50 mm3 or less. Further studies with BOPP (tetrakis-carborane carboxylate ester of 2,4-bis-(a, b-dihydroxyethyl)-deutero-porphyrin IX) showed that when this porphyrin was injected 5-7 days before BPA, and the animals were sacrificed 60 min after the i.p. injection of BPA, a significant increase in boron uptake by the tumor was found (45-38 ppm with both compounds vs. 20 ppm with BPA alone). The application of BNCT using the combination of boron compounds showed a 100% of complete remission in tumors with initial volumes under 50 mm3. Dogs suffer spontaneous UTC, with a similar biological behavior to the human tumor, and a selective uptake of BPA. These results open the possibility of applying BNCT to UTC

Effects of 2-APB on store-operated Ca2+ channel currents of hepatocytes after hepatic ischemia/reperfusion injury in rats / 华中科技大学学报（医学）（英德文版）

The effects of hepatic ischemia/reperfusion (1/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (Isoc) in isolated rat hepatocytes after hepatic ischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 micromol/L,) produced a concentration-dependent decrease of Isoc with IC50 value of 64. 63 +/- 10.56 micromol/L, (n = 8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.

Influence of different calcium and boron salts on cell wall degradation of melon plants caused by erwinia carotovora var. carotovora

The effect of different 10-6-103 M calcium nitrate, calcium sulfate, calcium chloride, calcium hypophosphate or calcium carbonate as well as 10-100 ppm sodium tetraborate on the activity of cellulase, pectinase, cell wall degrading enzymes and proteases was studied applying the colorimetric methods of assay. The results showed that raising the salts concentration was accompanied by a concomitant reduction in the activity of such enzymes. Furthermore, calcium nitrate, calcium sulfate and calcium hypophosphate were more effective inhibitors than the other test salts. In the meantime, using mixtures of calcium salts and sodium tetraborate revealed that borate + calcium nitrate or borate + calcium sulfate were more effective than the other mixtures

Reducing the severity of bacterial soft rot of melon by applying calcium and boron mixtures

Applying borate + calcium nitrate + calcium sulfate in moderate concentrations [in vitro or in vivo] definitely attenuated the extracellular cell wall degrading enzymes produced by Erwinia carotovora var. Carotovora. The obvious decrease in the activities may be associated with the presence of both calcium and borate. The results indicated that the disease severity was reduced to the maximum value of 10% by using 5 x 104 M borate + 105 M calcium sulfate plus 10-5 M calcium nitrate. This effective mixture significantly increased radicle and hypocotyl lengths and decreased the fresh of the whole seedling, whereas the dry weights were insignificantly affected. The results indicated that the average pH remained close to alkalinity by the end of the incubation period. The resistance of melon seedling may be attributed to the presence of high levels of calcium which form calcium complex with pectic substances rendering the resistance to hydrolysis