Developmental α2-adrenergic regulation of noradrenergic synaptic facilitation at cerebellar GABAergic synapses.

In the central nervous system, the normal development of neuronal circuits requires adequate temporal activation of receptors for individual neurotransmitters. Previous studies have demonstrated that α2-adrenoceptor (α2-AR) activation eliminates spontaneous action potentials of interneurons in the cerebellar molecular layer (MLIs) and subsequently reduces the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in Purkinje cells (PCs) after the second postnatal week. The magnitude of the α2-adrenergic reduction in sIPSC frequency is enhanced during the third postnatal week because of an increase in firing-derived sIPSCs. However, little is known about the effects of α2-AR activation by noradrenaline (NA) on cerebellar GABAergic synaptic transmission that is accompanied by the activation of other AR subtypes, α1- and ß-ARs. Here, we developmentally examined the roles of α2-AR activation in the noradrenergic facilitation of sIPSCs in cerebellar PCs. Until the second postnatal week, when substantial inhibitory effects of α2-ARs are absent, NA potentiated sIPSCs and maintained the increased sIPSC frequency, suggesting that NA causes long-lasting facilitation of GABAergic synaptic transmission through α1- and ß-AR activation. After the second postnatal week, NA transiently increased the sIPSC frequency, whereas blocking α2-ARs sustained the noradrenergic sIPSC facilitation and increase in the firing rate of MLIs, suggesting that α2-AR activation suppresses the noradrenergic facilitation of GABAergic synaptic transmission. The simultaneous activation of α1- and ß-ARs by their specific agonists mimicked the persistent facilitation of sIPSC frequency, which required extracellular signal-regulated kinase 1/2 activation. These findings indicate that NA acts as a neurotrophic factor that strengthens GABAergic synaptic transmission in the developing cerebellar cortex and that α2-ARs temporally restrain the noradrenergic facilitation of sIPSCs after GABAergic synaptogenesis.

Developmental enhancement of alpha2-adrenoceptor-mediated suppression of inhibitory synaptic transmission onto mouse cerebellar Purkinje cells.

Noradrenaline (NA) modulates glutamatergic and GABAergic transmission in various areas of the brain. It is reported that some alpha2-adrenoceptor subtypes are expressed in the cerebellar cortex and alpha2-adrenoceptors may play a role in motor coordination. Our previous study demonstrated that the selective alpha2-adrenoceptor agonist clonidine partially depresses spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse cerebellar Purkinje cells (PCs). Here we found that the inhibitory effect of clonidine on sIPSCs was enhanced during postnatal development. The activation of alpha2-adrenoceptors by clonidine did not affect sIPSCs in PCs at postnatal days (P) 8-10, when PCs showed a few sIPSCs and interneurons in the molecular layer (MLIs) did not cause action potential (AP). In the second postnatal week, the frequency of sIPSCs increased temporarily and reached a plateau at P14. By contrast, MLIs began to fire at P11 with the firing rate gradually increasing thereafter and reaching a plateau at P21. In parallel with this rise in the rate of firing, the magnitude of the clonidine-mediated inhibition of sIPSCs increased during postnatal development. Furthermore, the magnitude of the clonidine-mediated firing suppression in MLIs, which seemed to be mediated by a reduction in amplitude of the hyperpolarization-activated nonselective cation current, I(h), was constant across development. Both alpha2A- and alpha2B-, but not alpha2C-, adrenoceptors were strongly expressed in MLIs at P13, and P31. Therefore, the developmental enhancement of the clonidine-mediated inhibition of sIPSCs is attributed to an age-dependent increase in AP-derived sIPSCs, which can be blocked by clonidine. Thus, presynaptic activation of alpha2-adrenoceptors inhibits cerebellar inhibitory synaptic transmission after the second postnatal week, leading to a restriction of NA signaling, which is mainly mediated by alpha1- and beta2-adrenoceptors in the adult cerebellar neuronal circuit.

Adjuvant chemotherapy in stage I uterine endometrial carcinoma.

OBJECTIVE: We have assessed prognostic factors and the efficacy of adjuvant chemotherapy in stage I uterine endometrial carcinoma. METHODS: 251 primary surgically treated stage I patients were studied. Prognostic factors were evaluated and 5-year and 10-year survival rates were compared in patients with lymph-vascular space invasion to investigate whether adjuvant chemotherapy improves survival. RESULTS: The overall 5-year and 10-year survival rates were 94% and 93%. Multivariate analysis indicates that lymph-vascular space invasion is the most significant prognostic factor in both 5- and 10-year survival rates (P<0.001 at both times) and stage/depth of invasion is significant for the 10-year survival rate (P=0.04). Of 54 patients with lymph-vascular space invasion, statistically significant differences were observed in 10-year survival rate (P=0.02) between patients who had surgery followed by adjuvant chemotherapy (n=23) and patients who had surgery alone (n=31). Toxicities were mild to moderate (30%). CONCLUSIONS: The clinical importance of lymph-vascular space invasion and the efficacy of adjuvant chemotherapy were confirmed. This observation warrants a larger comparative study with adjuvant chemotherapy.

GABA(B) receptor activation enhances mGluR-mediated responses at cerebellar excitatory synapses.

Metabotropic gamma-aminobutyric acid type B (GABAB) and glutamate receptors (mGluRs) are postsynaptically co-expressed at cerebellar parallel fiber (PF)-Purkinje cell (PC) excitatory synapses, but their functional interactions are unclear. We found that mGluR1 agonist-induced currents and [Ca2+]i increases in PCs were enhanced following co-activation of GABAB receptors. A GABAB antagonist and a G-protein uncoupler suppressed these effects. Low-concentration baclofen, a GABAB agonist, augmented mGluR1-mediated excitatory synaptic current produced by stimulating PFs. These results indicate that postsynaptic GABAB receptors functionally interact with mGluR1 and enhance mGluR1-mediated excitatory transmission at PF-PC synapses. The interaction between the two types of metabotropic receptors provides a likely mechanism for regulating cerebellar synaptic plasticity.

Impaired delay but normal trace eyeblink conditioning in PLCbeta4 mutant mice.

To elucidate the functional role of phospholipase Cbeta4 (PLCbeta4), which is highly expressed in the Purkinje cells of the rostral cerebellum, cerebellar long-term depression (LTD) and delay and trace eyeblink conditioning were investigated in PLCbeta4-deficient mice. Rostral cerebellar LTD and delay eyeblink conditioning were severely impaired, whereas trace eyeblink conditioning was not. These results indicate that PLCbeta4 is essential for LTD in the rostral cerebellum and delay conditioning, but not trace conditioning. Rostral cerebellar LTD may be required as a neural substrate for delay conditioning, but is not required for trace conditioning.

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation.

Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells.

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.

Identification of N-acetyltransferase 2 and CYP2C19 genotypes for hair, buccal cell swabs, or fingernails compared with blood.

Genotyping of polymorphic drug metabolizing enzymes may be useful to estimate the blood concentration, efficacy, and toxicity of drugs before administration. Blood samples are most generally used for genotyping; however, sampling is invasive and complicated by handling and transport. Therefore, the authors developed genotyping methods using nonblood specimens, and then each genotype was compared with that from blood. Healthy Japanese volunteers provided hairs (n = 50), buccal cell swabs (n = 50), and fingernails (n = 30) for N-acetyltransferase 2 and CYP2C19 genotyping. Recovery of genomic DNA from each nonblood specimen was lower than that from 0.5 mL blood. Using a modification of the DNA extraction and polymerase chain reaction amplification method, genotypes were diagnosed without failure, even for those with very low levels of DNA. Both genotypes from these specimens completely matched the genotypes from the blood of the same subject. These nonblood specimens can be convenient, accessible, and economical alternatives to blood as a source of DNA for genotyping.

Expression of vascular endothelial growth factors (VEGF-A/VEGF-1 and VEGF-C/VEGF-2) in postmenopausal uterine endometrial carcinoma.

OBJECTIVE: We investigated the expression of two angiogenic vascular endothelial growth factors, VEGF-A/VEGF-1 and VEGF-C/VEGF-2, in 228 cases of uterine endometrial carcinomas from postmenopausal patients to evaluate the correlation with histopathologic features and clinical outcome. METHODS: Immunohistochemistry was used to assess VEGF-A/VEGF-1 and VEGF-C/VEGF-2 expression in 228 primary surgically treated cases of postmenopausal endometrial carcinomas and the results were statistically analyzed in relation to vascular invasion, depth of invasion (myometrial vs serosal-parametrial invasion), lymphatic vessel invasion, lymph node metastasis, disease-free 5-year survival rate (DF5YR), and disease-free 10 year-survival rate (DF10YR). RESULTS: The results of univariate analysis showed that VEGF-A/VEGF-1 and VEGF-C/VEGF-2 expression correlated with vascular invasion (P < 0.0001, P = 0.0006), depth of invasion (P = 0.0004, P = 0.043), lymphatic vessel invasion (P = 0.021, P < 0.0001), lymph node metastasis (P = 0.0017, P = 0.0008), DF5YR (P = 0.0081, P = 0.0002), and DF10YR (P = 0.0077, P = 0.0001). Multivariate analysis showed that lymph node metastasis (P = 0.0017, P = 0.0008), parametrial-serosal invasion (P < 0.0001, P < 0.0001), and VEGF-C/VEGF-2-positive status (P = 0.03, P = 0.01) were significant factors in DF5YR and DF10YR. CONCLUSIONS: We conclude that VEGF-A/VEGF-1 and VEGF-C/VEGF-2 expression was predictive of these histopathologic features of endometrial carcinoma and clinical outcome.

Sapecin B alters kinetic properties of rapidly inactivating K(+) channels in rat pituitary GH(3) cells.

Sapecin B is an antibacterial protein isolated and purified from culture medium of the embryonic cell line derived from the flesh fly (Sarcophaga peregrina). It has structural similarities to the scorpion toxin charybdotoxin (CTX). We have first detailed the effects of the newly described toxin (sapecin B) on the gating kinetics of the 4-aminopyridine-sensitive, rapidly inactivating K(+) current in rat pituitary GH(3) cells in order to investigate this protein's site of action, with whole-cell voltage-clamp methods. We have found that sapecin B alters the kinetics of activation and deactivation whereas there was no effect on the inactivation process. None of the effects of sapecin B was voltage dependent. In addition, sapecin B reduced whole-cell conductance. We suggest that the toxin may be ineffective against the voltage-sensitive segment, as well as the N-terminal and C-terminal domains, and CTX and sapecin B probably may have different binding sites.

Examination of microgravity effects on spontaneous Ca2+ oscillations in AtT20 pituitary cells using heavy water.

Effects of heavy water (D2O) on various organisms have been extensively studied and a majority of D2O actions were generally ascribed to the viscosity (1.23 times of H2O) and a larger inter-molecule force of D2O that may eventually alternate molecular structure of various enzymes and ion channels. It is reported that chronic application of D2O induces toxic effects and the 35% substitution of whole body water with D2O induced fatal effects in the mouse. Mitosis of a fertile egg of sea urchin was completely inhibited by 75% D2O but the paused segmentation was recovered after rinse of D2O. In addition, we also observed that neuronal development of the Lymnaea stagnalis was reversibly inhibited by D2O (M. Sakakibara, unpublished data). However, mechanism of the toxicity of D2O and the effects of D2O on cellular events have not been fully understood. The spontaneous oscillation in cytosolic free Ca2+ concentration is one of the typical physiological events in living secretory cells. We previously demonstrated that the Ca2+ oscillations are regulated by voltage-sensitive Ca2+ channels (VSCC), Ca2+ ATPases, and Ca(2+)-induced Ca2+ release from intracellular stores. To analyze the site(s) of action of D2O in the living cellular systems, the present study examined effects of D2O on the Ca2+ mobilization and resting membrane potentials in AtT20 mouse pituitary cells.

Localization of phospholipase Cbeta isozymes in the mouse cerebellum.

To elucidate the role of phospholipase Cbeta (PLCbeta) isozymes in the cerebellum, the distributions of PLCbeta3 and PLCbeta4 were examined in wild-type and PLCbeta4-deficient mutant mice using immunohistochemistry, and the functions were evaluated by measurement of type 1 metabotropic glutamate receptor (mGluR1)-mediated inward current and Ca(2+) mobilization. In wild-type mice, PLCbeta4 was distributed equally in both rostral and caudal cerebellum, while PLCbeta3 was enriched in the caudal versus the rostral cerebellum. In PLCbeta4-deficient mice, there was no measurable inward current or intracellular Ca(2+) elevation in the rostral cerebellum, whereas small responses were observed in the caudal cerebellum. In wild-type mice, the inward current was observed only following the release of caged GTPgammaS, not caged IP(3). These results suggest that the signal transduction machinery, including receptors, G-proteins, PLCbeta3, PLCbeta4, and effectors, form a functional unit, and the deletion of PLCbeta4 alters this unit, markedly changing signal transduction efficacy.

[Clinical outcome of an anticancer agent screening test with primary culture cells].

Cancer cells are tested for sensitivity to anticancer agents before the drugs are used for treatment in clinical settings, but the results of these tests have not always been reported fully. Since 1973, one of the authors (Hirono) has been performing sensitivity tests of anticancer drugs by using primary culture cells derived from human cancer tissue, taking into account in vivo status and taking great care to reduce the number of procedural errors. From 1991, flow cytometry has also been adopted for use in anticancer drug sensitivity tests. In the present study, the outcomes of cancer patients were compared by dividing them into a group treated only with anticancer agents to which cancer cells had responded in sensitivity tests, and a group treated with other drugs. The subjects consisted of 132 patients with endometrial cancer stage III (n = 26) and IV (n = 10), ovarian cancer stage III (n = 26) and IV (n = 7), peritonitis carcinomatosa (n = 18), and suspected advanced ovarian cancer (n = 45). There was a significant prolongation in median survival time and survival time among the non-survivors according to the results of a Kaplan-Meier analysis. These findings suggest that sensitivity testing of cancer cells to anticancer drugs should be performed before the start of cancer chemotherapy in the clinical setting.

Prognostic factors relating to survival in uterine endometrioid carcinoma.

OBJECTIVE: Epidemiologic and clinicohistopathologic prognostic factors of uterine endometrioid carcinomas were analyzed. The association of estrogen related factors, focused on adenomyosis in the prognosis of endometrioid carcinomas was also examined. METHODS: Risk factors of surgically treated 286 patients with endometrioid carcinoma (Stage I-III) were statistically analyzed. RESULTS: Overall a recurrence-free 5-year survival rate was 81% (Stage I, 94%, Stage II, 71% and Stage III, 40%). Significant prognostic factors were lymph node metastases (P = 0.0035) and serosal/parametrial invasion (P = 0.014) by multivariate analysis. Endometrioid carcinomas with co-existing adenomyosis tend to be associated with endometrial hyperplasia (P = 0.04, Fisher's exact test), diagnosed in less invasive status (myometrial invasion, P = 0.004 and serosal/parametrial invasion, P = 0.006) and therefore have a favorable prognosis (P = 0.01, log rank test). CONCLUSIONS: A favorable prognosis of endometrioid carcinomas with co-existing estrogen related factors (adenomyosis and endometrial hyperplasia) was suggested.

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.

Isolation and characterization of novel Chlamydomonas mutants that display phototaxis but not photophobic response.

The unicellular green alga Chlamydomonas displays two distinct kinds of behavioral response to light: phototaxis, in which cells swim toward or away from the light source under constant illumination; and photophobic responses (also called stop responses or photoshock responses), in which cells transiently convert their flagellar waveform and swim backward upon sudden increase in light intensity. It has been suggested that the two responses partly share a common signal transduction pathway, but exactly how the different responses are produced has not been established. In this study, to help understand the molecular and cellular mechanisms that bring about the photophobic response, we isolated novel mutants (ppr1, ppr2, ppr3, and ppr4) that do not show the photophobic response. Importantly, these mutants retain the ability to display phototaxis, with almost the same sensitivities as in the wild type cell. Demembranated and reactivated flagellar axonemes of the ppr mutants were found to convert the bending patterns depending on the Ca2+ concentration, indicating that the axonemal mechanism for waveform conversion required for the photophobic response was unaffected by the mutations. In addition, measurements of electric currents in cell suspensions showed that these mutants generate normal photoreceptor currents (PRC) upon photostimulation, suggesting that they retain the normal activity of photoreception and the ionic channels that produce PRCs. However, the all-or-none flagellar current (FC), a Ca2+ current generated by PRC-induced depolarization of flagellar membrane, was absent or seriously impaired in the mutants. These findings clearly indicate that the all-or-none FC is necessary for the photophobic response but not for phototaxis. The isolation of the four genetically independent ppr mutants suggests that the generation of the FC is based on multiple components that are not used in the mechanism for phototaxis, and implies that the Chlamydomonas flagellar membrane possesses a voltage-dependent Ca2+-channel specifically used for generation of photophobic responses.

Highly divergent actin expressed in a Chlamydomonas mutant lacking the conventional actin gene.

The Chlamydomonas mutant ida5 is deficient in the conventional actin gene and its axoneme lacks a subset of inner dynein arms that contain actin as a subunit. However, this mutant retains some other inner dynein arms because a novel protein (NAP) is expressed as a substitute for actin. In this study, we show by sequence analysis that NAP is identical to a putative actin-related protein, the cDNA sequence of which has recently been reported and shown to have 64% amino acid identity with conventional actin. A polyclonal antibody raised against a synthetic polypeptide corresponding to the NH2-terminal sequence of this protein specifically reacted with the spot corresponding to NAP in two-dimensional electrophoresis patterns. NAP apparently can substitute for conventional actin in some, but not all, cellular functions, and therefore can be regarded as a highly divergent actin. This unconventional actin appears to be expressed only when conventional actin is absent.

Phospholipase C-independent group I metabotropic glutamate receptor-mediated inward current in mouse purkinje cells.

The mGluR agonist 1S,3R-ACPD induces a potent excitatory inward current in various central neurons, but the underlying mechanism is not fully understood. Thus, we explored the signal transduction mechanism underlying the 1S,3R-ACPD-induced inward current in mouse cerebellar Purkinje cells. Iontophoretic application of 1S,3R-ACPD produced a group I mGluR antagonist-sensitive inward current. This current closely resembled a slow synaptic inward current evoked by repetitive stimulation of parallel fibers. Although phosphoinositide hydrolysis is shown to be coupled with group I mGluRs, we found that intracellular injections of various PLC inhibitors and an IP3 receptor antagonist heparin only partially inhibited the 1S, 3R-ACPD-induced current. Moreover, intracellular injection of the Ca2+ chelator BAPTA or a selective Na+/Ca2+ exchange inhibitor KB-R7943 affected slightly the inward current. In contrast, infusion of GDPbetaS and GTPgammaS markedly suppressed the 1S,3R-ACPD-induced current. These results suggest that activation of mGluR1 in mouse cerebellar Purkinje cells by 1S,3R-ACPD application or by repetitive stimulation of parallel fibres induces an inward current with a minor contribution from intracellular Ca2+ or Na+/Ca2+ exchange.