Vitamin D and intact PTH status in patients with hip fracture.

INTRODUCTION: The prevalence of hypovitaminosis D in patients with acute hip fracture was examined in a population on Sado Island in Japan. There were 85 cases of hip fracture among this population in 2004, giving an overall incidence of hip fracture of 121.4 per 100,000 population per year. This study included 50 of the 85 cases, and these cases were defined as the hip fracture group. Patients older than 70 years without established osteoporosis who were admitted to the hospital on the island during almost the same period for treatment of an orthopedic condition other than a hip fracture were defined as the control group. MATERIALS AND METHODS: The levels of serum 25-hydroxyvitamin D (25-OHD), intact parathyroid hormone (intact PTH), alkaline phosphatase (ALP), albumin, and the number of remaining teeth were examined in each group. In the hip fracture group, serum calcium, serum phosphorus, urine N-terminal cross-linking telopeptide of type I collagen (NTx), bone mineral density (BMD) of the nonfractured hip, the presence of a vertebral fracture on X-ray, severity of dementia, and physical activity level were also examined. RESULTS: Both the serum 25-OHD and serum albumin levels were significantly lower in patients with hip fracture than in controls, and the intact PTH level was significantly higher in patients with hip fracture. The number of remaining teeth was correlated with age, and was also significantly correlated with 25-OHD. In the hip fracture group, 62% of the subjects had hypovitaminosis D (25-OHD <20 ng/ml) and one-fifth of cases with hypovitaminosis D showed elevated PTH levels (>65 pg/ml). On the other hand, in the control group, hypovitaminosis D occurred in 18.9% of the subjects, and only one case showed elevated PTH. The serum 25-OHD level showed a decrease as the severity of dementia progressed and the activity level decreased. CONCLUSION: Our results indicate that about two-thirds (62%) of hip fracture patients had vitamin D insufficiency, suggesting that this condition may be closely associated with hip fracture in elderly people. Therefore, the serum 25-OHD level may be a useful index for the risk of hip fracture in elderly people.

PI3K-AKT pathway mediates growth and survival signals during development of fetal mouse lung.

We examined the roles of the PI3K-AKT signalling pathway in fetal lung development. By Western blotting, phosphorylated AKT (pAKT) was highly expressed in fetal days 12 and 14 with decreased expression thereafter. By immunohistochemistry, pAKT was expressed mainly in the respiratory epithelium of early fetal days. We examined the effects of fibroblast growth factor 1 (FGF1), PI3K inhibitors (LY294002 and wortmannin), MAPK inhibitor (PD98059) and both of FGF1 and each inhibitor on lung morphogenesis, BrdU incorporation and apoptosis. In the FGF1-treated explants, the number of terminal buds and BrdU-labelled cells increased significantly, while the LY294002-, wortmannin-, PD98059-treated explants demonstrated obvious decreases. The effects by FGF1 were inhibited by LY294002, wortmannin and PD98059. Regardless of the presence of FGF1, the LY294002-, wortmannin- and PD98059-treated explants increased apoptosis revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay in the mesenchyme of the explants. At the same time, the effect of LY294002, wortmannin, PD98059 on expression of surfactant apoprotein C (SPC) were also studied. The LY294002 and wortmannin treatments showed decreased expression of SPC. These findings suggest that the PI3K-AKT signalling pathway plays a pivotal role in mouse lung development through various biological processes.

Sites and modes of action of proctolin and the FLP F2 on lobster cardiac muscle.

At the threshold concentration (1-10 pmol l(-1)), the neuropeptide hormones proctolin (PR) and the FLRFamide-like peptide (FLP) F(2) cause an increase in amplitude of electrically evoked contractions (each contraction is a brief tetanus) of lobster heart ostial muscle. At higher concentrations each peptide also induces an increase in tonus (contracture). The PR-induced contracture and augmentation of tetani are proportional to increases in [Ca2+]i. The rate of onset and recovery of peptide-induced effects on both tetani and contracture appeared to reduced by Ca2+ storage by the sarcoplasmic reticulum (SR). Enhanced tetani following a contracture may be due to enhanced voltage-gated Ca2+ current and sarcoplasmic reticular (SR) Ca2+ loading. The SR Ca2+ loading appears to be specific for PR and F2, since glutamic-acid-induced contractures are not followed by increased tetani. The prolonged elevation of [Ca2+]i during contracture causes a right-ward shift in the force-pCa curve indicating a decrease in myofibrillar sensitivity to Ca2+. Blocking voltage-gated Ca2+ channels with Cd2+, nifedipine or verapamil, while reducing tetani, does not prevent peptide-induced contracture and enhanced tetani. Opening SR Ca2+ channels and depleting SR Ca2+ with either caffeine or ryanodine blocked tetani but permitted accelerated peptide-induced contractures. We conclude that PR and F2 at low concentration enhance voltage-dependent Ca2+ induced Ca2+ release from the SR, while higher hormone levels directly gate Ca2+ entry across the sarcolemma.

Expression of epiregulin and amphiregulin in the rat ovary.

We have previously reported that the epidermal growth factor (EGF) family growth factor, epiregulin, is expressed in rat ovarian granulosa cells by induction with pregnant mare serum gonadotropin (PMSG). In this study, we report that amphiregulin, another member of the EGF family, was also induced in the rat ovary by gonadotropin treatment. Northern blot analysis revealed that PMSG treatment induced the expression of both epiregulin and amphiregulin mRNA after 24 h, but the expression then decreased 48 h after treatment. Further treatment with human chorionic gonadotropin (hCG) rapidly induced the expression of both epiregulin and amphiregulin genes and maximal levels were reached 4 h after hCG treatment. A marginal increase in amphiregulin mRNA levels was also observed 6 h after PMSG treatment. In situ hybridization revealed that epiregulin and amphiregulin mRNAs were localized in the granulosa cells of large antral follicles. These spatio-temporal expression patterns were similar to those of cyclo-oxygenase-2 (COX-2) and progesterone receptor (PR). In adult cycling rats, epiregulin and amphiregulin were strongly induced at 1800 and 2000 h on proestrus coinciding with the preovulatory LH surge. An in situ hybridization study also showed that epiregulin and amphiregulin mRNAs were detectable in the granulosa cells of preovulatory ovarian follicles at 2000 h on proestrus, where transcripts of COX-2 and PR were co-localized with those of epiregulin and amphiregulin. These observations suggested that the EGF family members, epiregulin and amphiregulin, may play a role in the ovulatory process of cycling rats as well as in the induction of ovulation in immature rats.

The steady-state force-Ca2+ relationship in intact lobster (Homarus americanus) cardiac muscle.

The heart of the decapod crustacean is activated by regular impulse bursts from the cardiac ganglion. The cardiac pump function depends on ganglionic burst frequency, burst duration, and burst impulse frequency. Here, we activated isolated lobster cardiac ostial muscle (Orbicularis ostii muscle, OOM) by stimulus trains in vitro in order to characterize the response of the contractile apparatus to [Ca2+]i. We employed stimulus trains that generate a steady state between the [Ca2+]i and force in order to estimate the Ca2+ sensitivity of myofilaments. Force and [Ca2+]i transients were simultaneously recorded using a silicon strain gauge and the fluorescence of iontophoretically microinjected fura-2 salt. We examined the effects of tetanus duration (TD), the interval between trains, and 6 microM cyclopiazonic acid, an inhibitor of the SR Ca2+ pump, on the steady-state force-[Ca2+]i relationship. The instantaneous force-[Ca2+]i relationships appeared sigmoidal (EC50 and Hill coefficient, 98.8+/-32.7 nM and 2.47+/-0.20, mean +/- SD, respectively), as did the curves superimposed after 500 ms following the start of stimulation, indicating that the force-[Ca2+]i relationship had reached a steady state at that time. Also, the maximum activated force (Fmax) was estimated using the steady-state force-[Ca2+]i relationship. Prolonged stimulus trains, decreasing the interval between recurrent trains from 5 to 2.5 s, and cyclopiazonic acid each increased the measured EC50 without changing Fmax. The EC50 correlated strongly with averaged [Ca2+]i over time. We conclude that the steady-state force-[Ca2+]i relationships in the OOM indicate cooperation between force generation and Ca2+ binding by the myofilaments. Our data also suggest the existence of a novel Ca2+-dependent mechanism which reduces Ca2+ sensitivity and accelerates relaxation of lobster cardiac muscle myofilaments.

Modulation of pulmonary neuroendocrine cells in idiopathic interstitial pneumonia.

In order to reveal modulation of the number of pulmonary neuroendocrine cells (PNEC) in interstitial lung diseases and to clarify significance of cell proliferation activity in occurrence of PNEC, we counted airway PNEC of the patients of idiopathic interstitial pneumonia, secondary interstitial pneumonia and control lungs, and compared the number of PNEC with airway Ki-67 labeling. The lung tissue samples were obtained by video-assisted thoracoscopic surgery from 22 patients with usual interstitial pneumonia (UIP), 7 with non-specific interstitial pneumonia (NSIP), 8 with chronic hypersensitivity pneumonia (CHP), 13 with collagen vascular disease (CVD), and were compared with age-matched control lungs. The tissues were immunostained for chromogranin A and for Ki-67. Average incidence of bronchiolar PNEC in normal, UIP, NSIP, CHP, CVD lungs was 0.169%, 0.348%, 0.326%, 0.175% and 0.201%, respectively, and average Ki-67 labeling index in them was 0.241%, 1.186%, 1.605%, 1.058%, and 2.353%, respectively. And, in UIP lungs, PNEC incidence or Ki-67 labeling index was different according to pathological lesions. Thus, PNEC increase in the bronchiole of UIP, and the incidence of PNEC varies according to degree of activity of epithelial cell proliferation probably related to epithelial cell injury. Moreover, enhanced expression of human homolog of achaete-scute complex (hASH1) mRNA in UIP lungs suggests that hASH1 could play roles in the regulation of PNEC.

Excitation-contraction coupling in cardiac muscle of lobster (Homarus americanus); the role of the sarcolemma and sarcoplasmic reticulum.

The T-tubules and sarcoplasmic reticulum (SR) serving excitation-contraction (EC) coupling in lobster (Homarus americanus) cardiac muscle are similar to those in mammalian myocardium. Tetanic contraction is elicited by a burst of action potentials from the cardiac ganglion. In this study we evaluated the roles of the sarcolemma and SR in EC coupling of the ostial valve muscle (orbicularis ostii m. or OOM) of lobster heart. The OOM was mounted in a bath with saline on a microscope stage; force was measured by strain gauge. [Ca2+]i was measured using iontophoretically micro-injected fura-2 salt. Peak [Ca+]i, peak tetanic force and time to peak [Ca2+]i increased with that of stimulus train duration (TD), to a maximum at a TD of 500 ms. Force increased with [Ca2+]. Cd2+ reduced force by 90%; ryanodine and caffeine reduced tetanic [Ca2+]i transients by 80% and 70%, and force by 90% and 80%, respectively. Ryanodine, caffeine and cyclopiazonic acid slowed the decline of [Ca2+]i and force during relaxation. Relaxation required [Na+]o. The rate of decline of [Ca2+]i appeared to be a sigmoidal function of the [Ca2+]i and increased for any [Ca2+]i with TD. Inactivity slowed relaxation of force; stimulation accelerated relaxation. These data suggest important contributions of Ca2+ transport both across the sarcolemma and across the SR membrane during EC-coupling of lobster cardiac muscle, while average cytosolic [Ca2+]i regulates the rate of [Ca2+]i elimination during relaxation.

Molecular cloning of natriuretic peptide receptor A from bullfrog (Rana catesbeiana) brain and its functional expression.

A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.

Cloning and characterization of gonadotropin-inducible ovarian transcription factors (GIOT1 and -2) that are novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family.

Gonadotropins are essential for ovarian follicular development and differentiation. To identify genes that are rapidly induced by gonadotropin in the immature rat ovary, ovarian genes were screened by a subtraction cloning procedure. cDNA clones encoding novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and 2), were identified. Two isoforms of GIOT2 (GIOT2 alpha and 2 beta), which are probably produced by alternative splicing, also exist. Nucleotide sequence analysis revealed that GIOT1, but not GIOT2, contains the krüppel-associated box-A domain at the NH(2) terminus. RNA analyses revealed that these mRNAs were rapidly and temporarily induced by gonadotropins in the rat testis as well as in the ovary. In situ hybridization study revealed that expression of GIOT1 was induced in theca interna cells in the ovary and Leydig cells in the testis. Interestingly, the gene expression of GIOT1 is restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 gene is expressed ubiquitously. A functional analysis of GIOT1 and -2 by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but not GIOT2, is a transcriptional repressor and that the krüppel-associated box-A domain of GIOT1 is responsible for the transcriptional repressor activity. A GAL4-based yeast two-hybrid system was also used to identify proteins that interact with the rat GIOT1. We cloned genes encoding rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta, both of which are transcription-regulatory proteins. Interaction of these proteins with GIOT1 was directly demonstrated by GST pull-down assay. Our data strongly suggest that GIOT1 may function as a novel transcriptional repressor by working with rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta proteins and may play a significant role at the transcription level in the folliculogenesis.

Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters.

We examine the cell proliferation activity and expression of cyclin-dependent kinase inhibitors of the Cip/Kip family, p21Cip1, p27Kip1 and p57Kip2, in foetal hamster lungs to determine the expression patterns of the cyclin-dependent kinase inhibitors and to clarify the relationship between expression of the cyclin-dependent kinase inhibitors and lung development. Foetal hamster lungs on gestational days 12.5-16 (the day of birth) and adult lungs were fixed in 4% paraformaldehyde. Frozen sections were immunostained for the cyclin-dependent kinase inhibitors, and examined by immunostaining for Ki-67 and bromodeoxyuridine to determine the proliferation activity of the foetal lungs. During the foetal period, cell proliferation activity, as analysed by Ki-67 or bromodeoxyuridine labelling, decreased with development of the lung. In contrast to the gradual decrease of cell proliferation activity, cells with p27Kip1 immunoreactivity increased with development. On the other hand, p21Cip1-positive cells were most prominent around gestational day 14.5, while after birth positive cells decreased markedly. A few p57Kip2-positive cells were detected in the bronchiolar epithelium on gestational day 14.5. Western blotting analyses confirmed these immunostaining patterns. Thus, the levels of the cyclin-dependent kinase inhibitors of the Cip/Kip family are modulated in the lungs during the foetal period, and each shows a unique expression pattern. The cyclin-dependent kinase inhibitors may play roles not only in regulating cell proliferation activity but also in regulating other functions such as differentiation in the lung during the foetal period.

High cyclin E and low p27/Kip1 expressions are potentially poor prognostic factors in lung adenocarcinoma patients.

Cyclin E is an important regulator of entry into the S phase of the cell cycle. p27/Kip1 (p27) binds to cyclin E/Cdk2 complex and negatively regulates cell proliferation. We immunohistochemically examined the expression of cyclin E and p27 in 98 cases of resected lung adenocarcinoma to evaluate the prognostic significance of cyclin E and p27. Cyclin E was expressed in 16 cases (16%), and p27 was expressed in 41 cases (42%). Using Kaplan-Meier survival analysis, patients with cyclin E positive (P=0.0017) and p27 negative (P=0.011), both individually and in combination (P<0.0001), had a worse prognosis. We also analyzed the relationship of these findings to clinicopathological parameters, which revealed that cyclin E-positive, p27-negative cases had a higher Ki67 expression (P=0.012) and a higher rate of lymph node metastasis (P=0.0078) than other groups. Our results suggested that cyclin E over expression, in association with p27 reduction in particular, may potentially be a poor prognostic factor in lung adenocarcinoma patients. However, to verify the prognostic significance of these factors, a multivariate analysis of a larger number of patients should be undertaken.

Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin.

Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.

The role of preoptic area and anterior hypothalamus and median raphe nucleus on thermoregulatory system in freely moving rats.

To clarify the role of the preoptic area and anterior hypothalamus (PO/AH) on thermoregulatory system and the effects of serotonergic innervation from the median raphe nucleus (MRN) on body temperature (Tb), we perfused tetrodotoxin (TTX) solution into the PO/AH or MRN by using a microdialysis technique at different ambient temperatures (5, 23 and 35 degrees C) in freely moving rats. Tb was continuously monitored by using a telemetry system. In the MRN, perfusion of TTX solution induced significant hypothermia in the normal environment, a greater decrease in Tb during cold exposure and had no effect on Tb during heat exposure. In the PO/AH, perfusion of TTX solution induced significant hyperthermia in normal environment, a greater increase in Tb during heat exposure and had no effect on Tb during cold exposure. Our results indicate that the PO/AH regulates mainly heat loss or inhibits the loci regulating heat production. Furthermore, heat production appears to be regulated by other loci receiving serotonergic innervation from the MRN.

Caspase activity in newt spermatogonial apoptosis induced by prolactin and cycloheximide.

We previously showed in vivo and in vitro, that among the spermatogenic stages of the newt, prolactin (PRL) induces apoptosis specifically in the penultimate stage of secondary spermatogonia. In the current report, we demonstrate in vitro that cycloheximide (CHX), an inhibitor of protein synthesis, induces morphological apoptotic changes similar to those caused by PRL, such as chromatin condensation and apoptotic body formation. Next, we found that Z-VAD-fmk, an inhibitor of various caspases, suppressed the apoptosis induced by PRL and CHX, but ICE inhibitor Ac-YVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO did not. As high caspase activity was present in extracts of testes treated with CHX, we suggest that an unidentified caspase induces the morphological changes of apoptosis in newt spermatogonia.

Cold suppression of follicle-stimulating hormone activity on proliferation and survival of newt spermatogonia.

In newts elevated titers of plasma prolactin (PRL), induced by low temperature, cause apoptosis in the penultimatemitotic stage of spermatogonia, and this cell death is suppressed by antiserum against newt PRL, but only during the initial 3 days of exposure (Yazawa et al., 1999). Thus, factors other than PRL must be involved in spermatogonial death. Follicle-stimulating hormone (FSH) may be a plausible candidate. Accordingly, the current study examined the activityof FSH on the proliferation and survival of spermatogonia at low temperatures in vivo and in vitro. Porcine FSH (pFSH) administration in vivo inhibited spermatogonial death induced at 12 degrees C, but failed to do so at8 degrees C. Also pFSH promoted in vitro the proliferation of spermatogonia at 12 degrees C, but not at 8 degrees C. Furthermore,dibutyryl cyclic AMP stimulated in vitro DNA synthesis of secondary spermatogonia at 12 degrees C, but not at 8 degrees C. These different responses to temperatures were not caused by different levels of mRNA for the receptor of follicle-stimulating hormone, the numberof FSH binding sites, or FSH binding affinity to its receptors in the testicular cells. Thus, the results indicate that a temperature-sensitive period exists duringthe postreceptor process and is responsible for thelack of response of newt testis to FSH at 8 degrees C.

Cloning and functional expression of an E box-binding protein from rat granulosa cells.

Ovarian granulosa cells undergo cell growth and cytodifferentiation during follicular maturation. In a number of tissues, the gene expression that is responsible for the cytodifferentiation is largely dependent on E box(es) located upstream of the responsible genes. In this study, we report on the cloning of cDNA(s) encoding E box (5'-CACGTG-3')-binding protein from a rat granulosa cell cDNA library using a yeast one-hybrid system. When multiple E box sequences were used as target, we obtained a positive clone that encodes the rat homologue of upstream stimulatory factor 2 (USF2). An analysis of the nucleotide sequence and its deduced amino acid sequence reveals that rat USF2 protein consists of 346 amino acid residues and belongs to the basic helix-loop-helix/leucine zipper protein family. Northern blot analysis shows that rat USF2 mRNA exists as multiple forms between 1.6 and 2.2 kilobases. The size of the cloned insert was identical to that of the transcript of maximal length. Electrophoretic mobility shift assays showed that in vitro-translated rat USF2 specifically binds to the E box. In addition, cotransfection experiments with luciferase-reporter constructs in HepG2 cells reveal that the overexpression of rat USF2 leads to an increase of luciferase activity in the E box sequence-dependent manner. Thus, we report molecular cloning, expression, and functional characterization of full-length rat USF2 cDNA.

Expression of cyclin-dependent kinase inhibitors in taste buds of mouse and hamster.

Taste buds are specialized epithelial cell clusters in the oral squamous cell epithelium. Although taste buds have been reported to renew rapidly, the mechanism of cell cycle control in these specialized structures remains unresolved. To clarify the cell cycle status and role of cyclin-dependent kinase inhibitors (CDKI) for cell cycle control in the taste buds, we analyzed cell proliferation activity using bromodeoxyuridine (BrdU) and Ki-67 immunostainings and the expression of the Cip/Kip family of CDKI (p21Cip1, p27Kip1, and p57Kip2) in the circumvallate papillae of mouse and hamster. BrdU-positive cells were detected in the basal layer of the oral epithelium. In the taste buds, Ki-67-positive cells were seen in the basal area, with only a very few positive cells in the taste buds. Both p21Cip1 and p27Kip1 positive cells were seen in the suprabasal layer of the non-gustatory oral epithelium. In the taste buds, stronger p27Kip1 staining was detected than in the non-gustatory epithelium. Western blotting analysis revealed that p27Kip1 was abundant in the mucosal tissues from circumvallate papillae. Thus, our study suggests that the taste bud cells except for basal cells are post-mitotic cells and that the cell cycle arrest associated with taste bud cell differentiation could be regulated predominantly by p27Kip1.

Significance of proneural basic helix-loop-helix transcription factors in neuroendocrine differentiation of fetal lung epithelial cells and lung carcinoma cells.

In this brief review article, we describe how cell fate determination by which the airway epithelial cells become neuroendocrine or non-neuroendocrine is regulated by a network of basic helix-loop-helix transcription (bHLH) factors in a similar manner to neuronal differentiation, and how this system could work to determine cell differentiation of human lung carcinomas. Immunohistochemical studies reveal that mammalina achaete-scute complex homologue (Mash)1 is expressed in pulmonary neuroendocrine cells (PNEC), while hairy and Enhancer of split (Hes)1 is expressed in pulmonary non-neuroendocrine cells (non-PNEC). Studies using gene-deficient mice for the bHLH factors revealed that in Mash1 homozygous null mice no PNEC are detected, while PNEC increase markedly in Hes1 homozygous null mice. These observations suggest that Mash1 is an essential positive factor for neuroendocrine differentiation of lung epithelium, and that Hes1 is one of the repressive factors for neuroendocrine differentiation. Moreover, immunohistochemical studies revealed that Notch receptors are detected in non-PNEC, and thus the Notch signalling pathway could play a role in the determination of airway epithelial cell differentiation. In human lung carcinomas, a similar bHLH network should operate to determine cell differentiation phenotypes. Generally, expression of the human homologue of Mash1 (HASH1) is detected in small cell carcinoma and carcinoids, while Hes1 seems to be expressed mainly in non-small cell carcinoma. Thus, proneuronal bHLH factors may play roles in cell fate determination of the airway epithelial system, and may regulate human airway epithelial cells in diseased conditions.

Spontaneous and repetitive cardiac slowdown in the freely moving spiny lobster, Panulirus japonicus.

The fluctuation of heartbeat interval was investigated to assess cardio-regulatory nervous function in freely moving spiny lobsters. This was performed by time series analysis of the heartbeat interval recorded from restrained animals, freely moving animals, and isolated hearts. The heart rate of freely moving animals exhibited on/off switching: i.e., an elevated and maintained rate was repetitively interrupted by periods of decreased rate. Each period was initiated by a sudden decrease in rate and was terminated by an exponential return to normal activity. In order to explain this characteristic change in heart rate, we have constructed a neurotransmitter release-reuptake model for such bi-stable activity of cardio-regulatory nerves. The model was successful in reproducing the characteristic observed fluctuation. In freely moving animals, the brain seems to regulate the heart through the inhibitory nerve in an "on/off" manner. In the hearts of restrained animals and isolated hearts, the heart rate exhibited white-noise like fluctuation. This implies that stress impairs the normal bi-stable regulatory mode.