Segregation of Ca2+ signaling in olfactory signal transduction.

Olfactory signal transduction is conducted through a cAMP-mediated second messenger cascade. The cytoplasmic Ca2+ concentration increases through the opening of CNG channels, a phenomenon that underlies two major functions, namely, signal boosting and olfactory adaptation. Signal boosting is achieved by an additional opening of the Ca2+-activated Cl- channel whereas adaptation is regulated by Ca2+ feedback to the CNG channel. Thus, the influx of Ca2+ and the resultant increase in cytoplasmic Ca2+ levels play seemingly opposing effects: increasing the current while reducing the current through adaptation. The two functions could be interpreted as compensating for each other. However, in real cells, both functions should be segregated. Ca2+ dynamics in olfactory cilia need to be directly measured, but technical difficulties accompanying the thin structure of olfactory cilia have prevented systematic analyses. In this study, using a combination of electrophysiology, local photolysis of caged cAMP, and Ca2+ imaging, we found that free Ca2+ in the local ciliary cytoplasm decreased along with a reduction in the current containing Ca2+-activated Cl- components returning to the basal level, whereas Ca2+-dependent adaptation persisted for a longer period. The activity of Cl- channels is highly likely to be regulated by the free Ca2+ that is present only immediately after the influx through the CNG channel, and an exclusive interaction between Ca2+ and Ca2+-binding proteins that mediate the adaptation may modulate the adaptation lifetime.

Adenocarcinoma in situ or early-stage cervical cancer is a risk factor for preterm delivery after cervical conization: a multicenter observational study.

OBJECTIVE: Pregnancy after conization is associated with a high risk of preterm delivery. However, because risk factors for preterm delivery after conization remain unknown, we conducted a multicenter observational study to investigate risk factors associated with preterm delivery. METHODS: We selected patients who had previously undergone conization and reviewed medical records from 18 hospitals in cooperation with Keio University School of Medicine between January 2013 and December 2019. Women were classified as nulliparous and primiparous, and a multiple logistic regression analysis was performed to evaluate the relative contributions of the various maternal risk factors for preterm delivery (i.e. delivery before 37 gestational weeks). RESULTS: Among 409 pregnant women after conization, 68 women delivered preterm (17%). The incidence of nulliparity (p = .014) was higher and a history of preterm delivery (p = .0010) was more common in the preterm delivery group than in the term delivery group. Furthermore, the proportion of women diagnosed with adenocarcinoma in situ (AIS) and cervical cancer in the preterm delivery group was higher than that in the term delivery group (p = .0099 and .0004, respectively). In multiple regression models in nulliparous women, cervical cancer or AIS (Odds ratio [OR]: 4.16, 95% CI: 1.26-13.68, p = .019) and a short cervix in the second trimester (OR: 13.41, 95% CI: 3.88-46.42, p < .0001) increased the risk of preterm delivery. Furthermore, a history of preterm delivery (OR: 7.35, 95% CI: 1.55-34.86, p = .012), cervical cancer or AIS (OR: 5.07, 95% CI: 1.24-20.73, p = .024), and a short cervix in the second trimester (OR: 4.29, 95% CI: 1.11-16.62, p = .035) increased the risk of preterm delivery in the multiple regression models in primiparous women. CONCLUSION: Pregnant women who previously underwent conization are at risk for preterm delivery. The histological type of AIS and cervical cancer was evaluated as a risk factor for preterm delivery. KEY MESSAGESPrior preterm delivery, presence of a short cervix, and cervical cancer or AIS were predictors of preterm delivery after conization.The depth of conization in cervical cancer or AIS group was significantly larger than that in the CIN group.

Validation of the FIGO 2018 staging system of cervical cancer: Retrospective analysis of FIGO 2009 stage IB1 cervical cancer with tumor under 2 cm.

AIM: The International Federation of Gynecology and Obstetrics (FIGO) revised the cervical cancer staging system in 2018. This study aims to validate the revised staging system in patients with tumors <2 cm in size who were classified as FIGO 2009 stage IB1. METHODS: We evaluated 62 women with stage IB1 cervical cancer (FIGO 2009) who underwent radical hysterectomy as the initial treatment between November 2004 and August 2018 in our institution. The patients with FIGO 2009 stage IB1 and tumors <2 cm in size were enrolled. We reclassified their stage according to the FIGO 2018 staging system and analyzed their clinicopathological data retrospectively. RESULTS: Twenty-five patients met the inclusion criteria. According to the FIGO 2018 classification, 9 (36.0%) patients were classified as stage IA, 13 (52.0%) as stage IB1, and 3 (12.0%) as stage IIIC, respectively. One (11.1%), six (46.2%), and three (100%) patients with lymphovascular space invasion were classified as stage IA, IB1, and IIIC, respectively. No significant differences were found in the 5-year overall survival or progression-free survival among the three stages. CONCLUSIONS: As many as 36.0% of patients classified as FIGO 2009 stage IB1 with a tumor <2 cm in size were classified as stage IA in the FIGO 2018 classification. For these cases, a treatment less invasive than radical hysterectomy or radiotherapy might be sufficient. Our results suggest that cervical cancer patients with tumors <2 cm should be carefully diagnosed by performing cervical conization and assessed the pathological findings before hysterectomy.

Suppression of olfactory signal transduction by insecticides.

2,4,6-Trichloroanisole (TCA) is a well-known, potent off-flavour compound present in various foods and beverages. TCA has been hypothesised to be a universal cause of flavour loss experienced in daily life. Here, however, we show that titres for the suppression of olfactory transducer channels caused by low-quality bananas are much higher than those for that caused by the TCA itself contained in the banana. We resurveyed other components of low-quality bananas and found that bananas also contain an insecticide (chlorpyrifos), and that it suppresses olfactory transducer channels. Other insecticides also suppressed olfactory transducer channels. Hence, even after passing safety examinations, certain insecticides may decrease the quality of foods and beverages by reducing their intrinsic scents.

Electrical properties of cells from human olfactory epithelium.

OBJECTIVE: The electrical properties of olfactory cells (OCs) are typically examined using animals such as newts, mice, and frogs, with few studies on human OCs. This study investigated the electrical properties of human cells from olfactory epithelium (hCOEs) obtained from subjects of olfactory epithelium showing no clinical symptoms during endoscopic sinus surgery. METHODS: hCOEs were isolated by collagenase treatment for whole-cell patch clamp recording. The identity of the cells was confirmed by immunohistochemistry with an antibody against olfactory maker protein. Under the voltage clamp with the whole-cell recording configuration, the voltage-gated currents of isolated hCOEs were recorded when the membrane potential was depolarized from a holding potential of -100 mV in a stepwise manner between -90 mV and + 40 mV. RESULTS: Only one of 14 hCOE samples expressed a transient inward current at the depolarizing voltage step that was activated by depolarization beyond -40 mV and reached a peak at -30 mV. Delayed and sustained outward currents (444 ± 106 pA at + 40 mV pulse; n = 20) were suppressed by tetraethyl ammonium (n = 3), which is consistent with the properties of newt OCs. CONCLUSIONS: Most hCOEs did not exhibit the transient inward current observed in animal models. These findings provide insight into the physiological basis of the unique aspects of human olfactory signal transduction.

Second messenger molecules have a limited spread in olfactory cilia.

Odorants are detected by olfactory receptors on the sensory cilia of olfactory receptor cells (ORCs). These cylindrical cilia have a diameters of 100-200 nm, within which the components required for signal transduction by the adenylyl cyclase-cAMP system are located. The kinetics of odorant responses are determined by the lifetimes of active proteins as well as the production, diffusion, and extrusion/degradation of second messenger molecules (cAMP and Ca2+). However, there is limited information about the molecular kinetics of ORC responses, mostly because of the technical limitations involved in studying such narrow spaces and fine structures. In this study, using a combination of electrophysiology, photolysis of caged substances, and spot UV laser stimulation, we show that second messenger molecules work only in the vicinity of their site of generation in the olfactory cilia. Such limited spreading clearly explains a unique feature of ORCs, namely, the integer multiple of unitary events that they display in low Ca2+ conditions. Although the small ORC uses cAMP and Ca2+ for various functions in different regions of the cell, these substances seem to operate only in the compartment that has been activated by the appropriate stimulus. We also show that these substances remain in the same vicinity for a long time. This enables the ORC to amplify the odorant signal and extend the lifetime of Ca2+-dependent adaptation. Cytoplasmic buffers and extrusion/degradation systems seem to play a crucial role in limiting molecular spreading. In addition, binding sites on the cytoplasmic surface of the plasma membrane may limit molecular diffusion in such a narrow space because of the high surface/volume ratio. Such efficient energy conversion may also be broadly used in other biological systems that have not yet been subjected to systematic experiments.

2,4,6-trichloroanisole is a potent suppressor of olfactory signal transduction.

We investigated the sensitivity of single olfactory receptor cells to 2,4,6-trichloroanisole (TCA), a compound known for causing cork taint in wines. Such off-flavors have been thought to originate from unpleasant odor qualities evoked by contaminants. However, we here show that TCA attenuates olfactory transduction by suppressing cyclic nucleotide-gated channels, without evoking odorant responses. Surprisingly, suppression was observed even at extremely low (i.e., attomolar) TCA concentrations. The high sensitivity to TCA was associated with temporal integration of the suppression effect. We confirmed that potent suppression by TCA and similar compounds was correlated with their lipophilicity, as quantified by the partition coefficient at octanol/water boundary (pH 7.4), suggesting that channel suppression is mediated by a partitioning of TCA into the lipid bilayer of plasma membranes. The rank order of suppression matched human recognition of off-flavors: TCA equivalent to 2,4,6-tribromoanisole, which is much greater than 2,4,6-trichlorophenol. Furthermore, TCA was detected in a wide variety of foods and beverages surveyed for odor losses. Our findings demonstrate a potential molecular mechanism for the reduction of flavor.

Suppression and recovery of voltage-gated currents after cocaine treatments of olfactory receptor cells.

OBJECTIVE: Cocaine (1-5% concentrations) is commonly used as a local anesthetic for the otorhinolaryngeal surgery of the nasal cavity. Recent reports indicate that some patients complain of olfactory deficits after surgery, and decreased olfaction is found in cocaine abusers. In spite of these reports, the effects of cocaine on the olfactory receptor cells are unknown. METHODS: Effect of cocaine was examined in olfactory receptor cells isolated from the newt. Under the voltage clamp with the whole-cell recording configuration, the voltage-gated currents were recorded when the membrane potential was depolarized from a holding potential of -100 mV in a step wise between -90 mV and +40 mV. RESULTS: When cocaine was applied by a puff pressure (5%) and the extracellular solution, the voltage-gated currents, including inward and outward components, were significantly reduced. The dose-suppression curves of cocaine for sodium and potassium currents could be fitted by the Hill equation. Half-blocking concentration of sodium and potassium currents were 43 µM and 557 µM; Hill coefficient was 1.1 and 0.9, respectively. CONCLUSION: This rapid and complete recovery from the suppression was confirmed even after the treatments with the high concentration cocaine. This fact implies that cocaine does not affect olfactory ability after locally high dose treatments of nasal cavity in surgical operation.

Recordings from cultured newt olfactory receptor cells.

Freshly dissociated olfactory receptor cells (ORCs) are commonly used in electrophysiological research investigations of the physicochemical mechanisms of olfactory signal transduction. Because the morphology of cultured cells clearly becomes worse over time, the ORCs are examined traditionally within several days after dissociation. However, there has been a major concern that cells are affected soon after dissociation. To gain a better understanding of the reliability of data obtained from solitary cells, we obtained electrical data during the lifetime of single ORCs dissociated from the newt. The time course for the deterioration could be revealed by monitoring the membrane properties during culture. Although the number of living cells that were identified by trypan blue extrusion declined day by day, the remaining cells retained morphology and their fundamental electrical features until day 19. In some cells, the cilia and dendrite were observed until day 21, and the bipolar morphology until day 31. The fundamental features of cell excitation were maintained during culture without showing remarkable changes when they retained morphological features. The results suggest that electrical properties of cells are almost unchanged within several days. Furthermore, the dissociated newt ORCs can be used for several weeks that are almost comparable to the intrinsic lifetime of the ORCs in vivo.

Intrinsic and extrinsic mechanisms control the termination of cortical interneuron migration.

During development, neurons migrate from their site of origin to their final destinations. Upon reaching this destination, the termination of their migration is crucial for building functional architectures such as laminated structures and nuclei. How this termination is regulated, however, is not clear. Here, we investigated the contribution of cell-intrinsic mechanisms and extrinsic factors. Using GAD67-GFP knock-in mice and in utero electroporation cell labeling, we visualized GABAergic neurons and analyzed their motility in vitro. We find that the motility of GABAergic neurons in cortical slices gradually decreases as development proceeds and is almost abolished by the end of the first postnatal week. Consistent with this, a reduction of embryonic interneuron motility occurred in dissociated cultures. This is in part due to cell-intrinsic mechanisms, as a reduction in motility is observed during long-term culturing on glial feeder cells. Cell-intrinsic regulation is further supported by observations that interneurons labeled in early stages migrated more actively than those labeled in late stages in the same cortical explant. We found evidence suggesting that upregulation of the potassium-chloride cotransporter KCC2 underlies this intrinsic regulation. Reduced motility is also observed when embryonic interneurons are plated on postnatal cortical feeder cells, suggesting extrinsic factors derived from the postnatal cortex too contribute to termination. These factors should include secreted molecules, as cultured postnatal cortical cells could exercise this effect without directly contacting the interneuron. These findings suggest that intrinsic mechanisms and extrinsic factors coordinate to reduce the motility of migrating neurons, thereby leading to the termination of migration.

Chemical structures of odorants that suppress ion channels in the olfactory receptor cell.

It has been proposed that odorant suppression of the cyclic nucleotide-gated (CNG) channel is responsible for olfactory masking. In this study, the effect of odorant chain length and functional group on this suppression was investigated. Because similar suppression has been observed for voltage-gated channels also, we used voltage-gated Na channels in the olfactory receptor cell as a tool for substance screening. These features were then re-examined using CNG channels. Interestingly, both CNG and Na channels were suppressed in a similar manner-carboxylic acids had little effect and suppression became stronger when the chain length of the alcohol or ester was increased. Degree of suppression was correlated with the distribution coefficients (Log D), irrespective of the molecules used. Results obtained here may provide information for the development of novel masking agents based on molecular architecture.

Expression of TGFß1 and its receptors is associated with biological features of ovarian cancer and sensitivity to paclitaxel/carboplatin.

It has been suggested that expression of TGFß1 and its receptors [TGFß receptor type I (TßRI) and TGFß receptor type II (TßRII)] may play a key role in the proliferation and progression of epithelial ovarian cancer. We investigated the biological significance of TGFß1 and its receptors, as well as their association with the tumor response to paclitaxel (PTX) and carboplatin (CBDCA). We studied 24 patients with ovarian cancer, primary peritoneal cancer, or fallopian tube cancer who had undergone surgery and chemotherapy with PTX and CBDCA. Tissues from the primary tumor were examined and the expression of TGFß1, TßRI, and TßRII mRNA was assessed by the RNase protection assay. It was found that TGFß1 mRNA expression was significantly lower in the tumors of patients who had optimal surgery than in the tumors of patients with suboptimal surgery. TGFß1 mRNA expression was also significantly lower in tumors with high sensitivity to PTX and CBDCA than in those with low sensitivity. TßRI mRNA expression was not associated with any clinicopathological factors. Expression of TßRII mRNA was significantly higher in clear cell adenocarcinoma and mucinous adenocarcinoma, while it was lower in serous adenocarcinoma and endometrioid adenocarcinoma. Moreover, it tended to be higher in early-stage tumors compared with advanced tumors. Among TGFß1, TßRI, and TßRII, expression of TGFß1 mRNA was most strongly associated with progression-free survival. When the prognosis of the patients with advanced cancer was compared on the basis of TGFß1 mRNA expression, those whose tumors showed low expression tended to have a better prognosis than those whose tumors showed high expression. It is suggested that TGFß1 mRNA expression is an indicator of tumor sensitivity to standard therapy with PTX and CBDCA, that it can identify biologically aggressive and highly malignant tumors and that it can predict the prognosis of patients with ovarian cancer.

Fibroma of the omentum resembling an ovarian tumor in the pelvis.

We would like to report the case of a patient with fibroma of the omentum that resembled an ovarian tumor in the pelvis. Since primary tumours of the omentum are rare, there is a paucity of information about the biology of such tumors in the basic texts and literature. An ultrasound examination of the patient revealed a mass, likely of ovarian origin, which consisted of liquid and solid components. It was suspected to be a malignant ovarian tumor. However, laparotomy demonstrated it was an omental tumor. This case shows that it can be difficult to pre-operatively diagnose omental fibromas because of their close resemblance to ovarian tumors.

Mechanism of olfactory masking in the sensory cilia.

Olfactory masking has been used to erase the unpleasant sensation in human cultures for a long period of history. Here, we show a positive correlation between the human masking and the odorant suppression of the transduction current through the cyclic nucleotide-gated (CNG) and Ca2+-activated Cl- (Cl(Ca)) channels. Channels in the olfactory cilia were activated with the cytoplasmic photolysis of caged compounds, and their sensitiveness to odorant suppression was measured with the whole cell patch clamp. When 16 different types of chemicals were applied to cells, cyclic AMP (cAMP)-induced responses (a mixture of CNG and Cl(Ca) currents) were suppressed widely with these substances, but with different sensitivities. Using the same chemicals, in parallel, we measured human olfactory masking with 6-rate scoring tests and saw a correlation coefficient of 0.81 with the channel block. Ringer's solution that was just preexposed to the odorant-containing air affected the cAMP-induced current of the single cell, suggesting that odorant suppression occurs after the evaporation and air/water partition of the odorant chemicals at the olfactory mucus. To investigate the contribution of Cl(Ca), the current was exclusively activated by using the ultraviolet photolysis of caged Ca, DM-nitrophen. With chemical stimuli, it was confirmed that Cl(Ca) channels were less sensitive to the odorant suppression. It is interpreted, however, that in the natural odorant response the Cl(Ca) is affected by the reduction of Ca2+ influx through the CNG channels as a secondary effect. Because the signal transmission between CNG and Cl(Ca) channels includes nonlinear signal-boosting process, CNG channel blockage leads to an amplified reduction in the net current. In addition, we mapped the distribution of the Cl(Ca) channel in living olfactory single cilium using a submicron local [Ca2+]i elevation with the laser photolysis. Cl(Ca) channels are expressed broadly along the cilia. We conclude that odorants regulate CNG level to express masking, and Cl(Ca) in the cilia carries out the signal amplification and reduction evenly spanning the entire cilia. The present findings may serve possible molecular architectures to design effective masking agents, targeting olfactory manipulation at the nano-scale ciliary membrane.

Distribution, amplification, and summation of cyclic nucleotide sensitivities within single olfactory sensory cilia.

Submicron local cAMP elevation was used to map the distribution of transduction channels in single olfactory cilia. After the fine fluorescent visualization of the cilium with the laser-scanning confocal microscope, the intraciliary cAMP was jumped locally with the laser beam that photolyzes cytoplasmic caged compounds. Simultaneously, cells' responses were obtained with the whole-cell patch clamp. Responses were observed anywhere within the cilia, showing the broad distribution of transduction channels. For odor detection, such distribution would be useful for expanding the available responding area to increase the quantum efficiency. Also, the stimulus onto only 1 microm region induced >100 pA response operated by >700-2300 channels, although only 1 pA is sufficient for olfactory cells to generate action potentials. The large local response indicates a presence of strong amplification achieved with a high-density distribution of the transduction channels for the local ciliary excitation.

A heavily pretreated patient with recurrent clear cell adenocarcinoma of the ovary in whom carcinomatous peritonitis was controlled successfully by salvage therapy with gemcitabine.

INTRODUCTION: Advanced clear cell adenocarcinoma of the ovary is a histologic type with an extremely poor prognosis. No reports have been published concerning useful drugs for salvage chemotherapy for this type of cancer. We performed salvage therapy with gemcitabine in a patient with multiple-drug- resistant, unresectable recurrent clear cell adenocarcinoma of the ovary and succeeded in stabilizing recurrent lesions and controlling carcinomatous peritonitis. CASE REPORT: A 55-year-old woman was in Stage IIIc of clear cell adenocarcinoma of the ovary. She had recurrent tumors after primary cytoreductive surgery, which were unresectable and also resistant to paclitaxel, carboplatin, irinotecan, and oral etoposide. After three courses of fourth-line chemotherapy with gemcitabine for the treatment of carcinomatous peritonitis and hepatic and splenic metastatic lesions, serum CA-125 and the severity of ascites showed marked decreases, and its efficacy for the hepatic and splenic metastatic lesions was classified as 5-month stable disease. The toxicity of this drug was in the acceptable range. CONCLUSION: Gemcitabine is also useful for heavily pretreated clear cell adenocarcinoma of the ovary. It is necessary to consider the use of drugs without cross resistance to platinum and taxanes in the selection of drugs for this cancer.

Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly.

Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein-coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca(2+) are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.

[Olfactory signal transduction: quantification of molecular chains held in a nano-scale structure].

Olfactory transduction is achieved by the G-protein-mediated molecular system. For a long period of time, research on the olfactory transduction system has been limited to qualitative descriptions, because of experimental difficulties accompanying use of diverse ligands, multiple receptors and the fine structure of the sensory cilia (200 nm diameter). However, recent works gradually overcame those difficulties to reveal the molecular systems within nano-scale biological structure. When the cAMP dynamics were estimated, the cAMP-production rate was on the order of 20,000/s/cilium at the maximum odorant stimulus. This number is much smaller than that known for other systems, and therefore it is suggested that the olfactory enzymes use low signal amplification. Apparently, signal transduction with a small number of molecules is achieved by the fine ciliary structure that has a high surface-volume ratio in which even a small change in the absolute number of molecules is reflected in a big change in concentration. In addition, the olfactory receptor cell has a unique and strong non-linear amplification detecting a slight change in the odorant dose, which is regulated by Ca2- that flows through the CNG channel; a cytoplasmic increase of Ca2- in turn activates excitatory Cl current to boost the net transduction current.