Cell Responses of the Ventrolateral Medulla to PAR1 Activation and Changes in Respiratory Rhythm in Newborn Rat En Bloc Brainstem-Spinal Cord Preparations.

Proteinase-activated receptor-1 (PAR1) is expressed in astrocytes of various brain regions, and its activation is involved in the modulation of neuronal activity. Here, we report effects of PAR1 selective agonist TFLLR on respiratory rhythm generation in brainstem-spinal cord preparations. Preparations were isolated from newborn rats (P0-P4) under deep isoflurane anesthesia and were transversely cut at the rostral medulla. Preparations were superfused with artificial cerebrospinal fluid (25-26 °C), and inspiratory C4 ventral root activity was monitored. The responses to TFLLR of cells close to the cut surface were detected by calcium imaging or membrane potential recordings. Application of 10 µM TFLLR (4 min) induced a rapid and transient increase of calcium signal in cells of the ventrolateral respiratory regions of the medulla. More than 88% of responding cells (223/254 cells from 13 preparations) were also activated by low (0.2 mM) K+ solution, suggesting that they were astrocytes. Immunohistochemical examination demonstrated that PAR1 was expressed on many astrocytes. Respiratory-related neurons in the medulla were transiently hyperpolarized (-1.8 mV) during 10 µM TFLLR application, followed by weak membrane depolarization after washout. C4 burst rate decreased transiently in response to application of TFLLR, followed by a slight increase. The inhibitory effect was partially blocked by 50 µM theophylline. In conclusion, activation of astrocytes via PAR1 resulted in a decrease of inspiratory C4 burst rate in association with transient hyperpolarization of respiratory-related neurons. After washout, slow and weak excitatory responses appeared. Adenosine may be partially involved in the inhibitory effect of PAR1 activation.

Minocycline prevents hypoxia-induced seizures.

Severe hypoxia induces seizures, which reduces ventilation and worsens the ictal state. It is a health threat to patients, particularly those with underlying hypoxic respiratory pathologies, which may be conducive to a sudden unexpected death in epilepsy (SUDEP). Recent studies provide evidence that brain microglia are involved with both respiratory and ictal processes. Here, we investigated the hypothesis that microglia could interact with hypoxia-induced seizures. To this end, we recorded electroencephalogram (EEG) and acute ventilatory responses to hypoxia (5% O2 in N2) in conscious, spontaneously breathing adult mice. We compared control vehicle pre-treated animals with those pre-treated with minocycline, an inhibitory modulator of microglial activation. First, we histologically confirmed that hypoxia activates microglia and that pre-treatment with minocycline blocks hypoxia-induced microglial activation. Then, we analyzed the effects of minocycline pre-treatment on ventilatory responses to hypoxia by plethysmography. Minocycline alone failed to affect respiratory variables in room air or the initial respiratory augmentation in hypoxia. The comparative results showed that hypoxia caused seizures, which were accompanied by the late phase ventilatory suppression in all but one minocycline pre-treated mouse. Compared to the vehicle pre-treated, the minocycline pre-treated mice showed a delayed occurrence of seizures. Further, minocycline pre-treated mice tended to resist post-ictal respiratory arrest. These results suggest that microglia are conducive to seizure activity in severe hypoxia. Thus, inhibition of microglial activation may help suppress or prevent hypoxia-induced ictal episodes.

Clinical application of long-read nanopore sequencing in a preimplantation genetic testing pre-clinical workup to identify the junction for complex Xq chromosome rearrangement-related disease.

OBJECTIVE: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders, such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult. METHODS: In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications. RESULTS: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination. CONCLUSION: Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements.

Role of microglia in blood pressure and respiratory responses to acute hypoxic exposure in rats.

Microglia modulate cardiorespiratory activities during chronic hypoxia. It has not been clarified whether microglia are involved in the cardiorespiratory responses to acute hypoxia. Here we investigated this issue by comparing cardiorespiratory responses to two levels of acute hypoxia (13% O2 for 4 min and 7% O2 for 5 min) in conscious unrestrained rats before and after systemic injection of minocycline (MINO), an inhibitor of microglia activation. MINO increased blood pressure but not lung ventilation in the control normoxic condition. Acute hypoxia stimulated cardiorespiratory responses in MINO-untreated rats. MINO failed to significantly affect the magnitude of hypoxia-induced blood pressure elevation. In contrast, MINO tended to suppress the ventilatory responses to hypoxia. We conclude that microglia differentially affect cardiorespiratory regulation depending on the level of blood oxygenation. Microglia suppressively contribute to blood pressure regulation in normoxia but help maintain ventilatory augmentation in hypoxia, which underscores the dichotomy of central regulatory pathways for both systems.

Novel oxygen sensing mechanism in the spinal cord involved in cardiorespiratory responses to hypoxia.

As blood oxygenation decreases (hypoxemia), mammals mount cardiorespiratory responses, increasing oxygen to vital organs. The carotid bodies are the primary oxygen chemoreceptors for breathing, but sympathetic-mediated cardiovascular responses to hypoxia persist in their absence, suggesting additional high-fidelity oxygen sensors. We show that spinal thoracic sympathetic preganglionic neurons are excited by hypoxia and silenced by hyperoxia, independent of surrounding astrocytes. These spinal oxygen sensors (SOS) enhance sympatho-respiratory activity induced by CNS asphyxia-like stimuli, suggesting they bestow a life-or-death advantage. Our data suggest the SOS use a mechanism involving neuronal nitric oxide synthase 1 (NOS1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We propose NOS1 serves as an oxygen-dependent sink for NADPH in hyperoxia. In hypoxia, NADPH catabolism by NOS1 decreases, increasing availability of NADPH to NOX and launching reactive oxygen species-dependent processes, including transient receptor potential channel activation. Equipped with this mechanism, SOS are likely broadly important for physiological regulation in chronic disease, spinal cord injury, and cardiorespiratory crisis.

Identification and phylogenetic analysis of Babesia parasites in domestic dogs in Nigeria.

The present study examined the presence of Babesia parasites in 104 domestic dogs in Nigeria. Sequentially, Babesia parasites infecting domestic dogs underwent genetic and phylogenetic analyses. The results of nested PCR based on the Piroplasmida 18S rRNA gene illustrated that 13.5% (14/104) of the samples were positive. The obtained positive samples determined the nucleotide sequences of the 18S rRNA genes. In the genetic and phylogenetic analyses, four of five nucleotide sequences were similar to Babesia canis rossi, and one sample exhibited a close similarity to a Babesia sp. isolated from a raccoon in Hokkaido, Japan. The present study revealed the widespread presence of B. canis rossi among domestic dogs in Nigeria.

Intrinsic properties and synaptic connectivity of Phox2b-expressing neurons in rat rostral parvocellular reticular formation.

The paired-like homeobox 2b gene (Phox2b) is critical for the development of the autonomic nervous system. We have previously demonstrated the distinct characteristics of Phox2b-expressing (Phox2b+) neurons in the reticular formation dorsal to the trigeminal motor nucleus (RdV), which are likely related to jaw movement regulation. In this study, we focused on Phox2b+ neurons in the rostral parvocellular reticular formation (rPCRt), a critical region for controlling orofacial functions, using 2-11-day-old Phox2b-EYFP rats. Most Phox2b+ rPCRt neurons were glutamatergic, but not GABAergic or glycinergic. Approximately 65 % of Phox2b+ rPCRt neurons fired at a low frequency, and approximately 24 % of Phox2b+ rPCRt neurons fired spontaneously, as opposed to Phox2b+ RdV neurons. Stimulation of the RdV evoked inward postsynaptic currents in more than 50 % of Phox2b+ rPCRt neurons, while only one Phox2b+ rPCRt neuron responded to stimulation of the nucleus of the solitary tract. Five of the 10 Phox2b+ neurons sent their axons that ramified within the trigeminal motor nucleus (MoV). Of these, the axons of the two neurons terminated within both the MoV and rPCRt. Our findings suggest that Phox2b+ rPCRt neurons have distinct electrophysiological and synaptic properties that may be involved in the motor control of feeding behavior.

Twin pregnancy with untyped Ehlers-Danlos syndrome requiring prompt genetic testing: A case report.

Ehlers-Danlos syndrome is a rare genetic disorder that presents with a variety of pathologies depending on the disease type. Among them, vascular Ehlers-Danlos syndrome requires extremely careful management as there have been many reports of fatal perinatal complications such as uterine rupture. Although hypermobile Ehlers-Danlos syndrome is less likely to cause fatal complications, symptoms such as arthralgia, hip dislocation, and depression may be seen throughout pregnancy. We report here a case of twin pregnancy in which Ehlers-Danlos syndrome was first suspected at 19 weeks of gestation. Vascular Ehlers-Danlos syndrome could not be ruled out based on family medical history, making it difficult to determine the perinatal management strategy. Prompt genetic testing did however rule out the vascular type and the patient was diagnosed with hypermobile Ehlers-Danlos syndrome from the clinical symptoms, enabling us to manage the pregnancy safely until 34 weeks of gestation.

Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat.

Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.

Aromatic interaction of hydantoin compounds leads to virucidal activities.

Virus inactivation or disinfection is the first line of protection against virus infection. Here, we report for the first time the virus inactivation (virucidal) activities of hydantoin and its derivative, 1-methylhydantoin against enveloped herpes simplex virus type-1. These hydantoin compounds showed favorable interaction with aromatic amino acids, similarly to arginine hydrochloride also exhibiting aromatic interaction and virucidal activities on the same virus. Among them, 1-methylhydantoin demonstrated a greater virucidal activity. Solubility measurements in organic solvents and salting-out salt solutions showed that 1-methylhydantoin is more hydrophobic than others, suggesting that the hydrophobic nature and aromatic interaction play a role in interaction with viral proteins and thereby virucidal activity.

Effects of temporary cessation of milking following intramammary cefazolin sodium infusion on residual antibiotic in lactating dairy cows.

The aims of studies were to estimate the withdrawal period of antibiotic from milk after the intramammary infusion of cefazolin sodium (CEZ) in cows with difficulties in frequent milk discharge due to disease such as teat injury. The period was compared among cows milked twice a day after 150 or 450 mg of CEZ were administered to all quarters (Study 1, 2) and the cows in which milking of front-right quarter was ceased for five days after administration of these infusions to only that quarter (Study 3). In Studies 1 and 2, the median of 17.66 µg/ml and 83.18 µg/ml of CEZ were detected in the samples of first milking after intramammary administration, respectively; however, there was no residual antibiotic by 72 hr in all cows. In Study 3, the median of 1.96 µg/ml of CEZ was detected in the sample after the resumption of milking at 120 hr, and the residual was eliminated by 174 hr. The withdrawal period may be prolonged by the cessation of milking after administration, and the period is the total time from cessation to 72 hr after the resumption of milking.

Validation of the bovine blood calcium checker as a rapid and simple measuring tool for the ionized calcium concentration in cattle.

Point-of-care (POC) devices that veterinary practitioners can use to easily and rapidly measure blood ionized calcium (iCa) levels in cows immediately after withdrawing a blood sample on the dairy farm are needed. Aims of present studies was to compare the commercially available ion-selective electrode handheld iCa meter (bovine blood iCa checker) with the benchtop blood gas analyzer GEM premier 3500 and handheld analyzer i-STAT 1. Sixty-two paired-point whole blood samples were obtained from three cows with hypocalcemia experimentally induced by Na2-EDTA infusion. Whole blood samples were also obtained from the 36 cows kept on a farm in field conditions. The results using the bovine blood iCa checker correlated with those using the GEM premier 3500 and i-STAT 1. Bovine blood iCa checker was "compatible" with the GEM premier 3500 and i-STAT 1 because the frequency of differences between the measurements within ± 20% of the mean were 100% (65/65, >75%) and 90.8% (59/65, >75%), respectively. In the field trial, the blood iCa concentration measured by the bovine blood Ca checker was significantly positively correlated with that measured by the i-STAT 1 portable analyzer. Bovine blood iCa checker was "compatible" with the i-STAT 1 because the frequency of differences between the measurements within ± 20% of the mean was 100% (36/36, >75%). Results from these findings, the bovine blood iCa checker may be applied as a simplified system to measure the iCa concentration in bovine whole blood.

Serum iron concentration is candidate inflammatory marker for respiratory diseases in beef cows.

We hypothesized that the serum iron (Fe) concentration in cows with respiratory diseases is a satisfactory substitute for major inflammatory markers such as haptoglobin (HPT) and serum amyloid A (SAA). Twenty Japanese Black cows aged 279.6 ± 120.0 days were enrolled, and divided into respiratory diseases and control groups based on the presence of clinical findings of respiratory diseases. As a result, area under the receiver operating characteristic curves for plasma HPT, SAA and serum Fe concentrations for respiratory disease-associated systemic inflammation were excellent, at 1.00, 0.96 and 0.97, respectively. Therefore we confirmed that the serum Fe concentration is a satisfactory substitute for HPT and SAA in beef cows with respiratory diseases.

Decreased content of ascorbic acid (vitamin C) in the brain of knockout mouse models of Na+,K+-ATPase-related neurologic disorders.

Na+,K+-ATPase is a crucial protein responsible for maintaining the electrochemical gradients across the cell membrane. The Na+,K+-ATPase is comprised of catalytic α, ß, and γ subunits. In adult brains, the α3 subunit, encoded by ATP1A3, is predominantly expressed in neurons, whereas the α2 subunit, encoded by ATP1A2, is expressed in glial cells. In foetal brains, the α2 is expressed in neurons as well. Mutations in α subunits cause a variety of neurologic disorders. Notably, the onset of symptoms in ATP1A2- and ATP1A3-related neurologic disorders is usually triggered by physiological or psychological stressors. To gain insight into the distinct roles of the α2 and α3 subunits in the developing foetal brain, whose developmental dysfunction may be a predisposing factor of neurologic disorders, we compared the phenotypes of mouse foetuses with double homozygous knockout of Atp1a2 and Atp1a3 (α2α3-dKO) to those with single knockout. The brain haemorrhage phenotype of α2α3-dKO was similar to that of homozygous knockout of the gene encoding ascorbic acid (ASC or vitamin C) transporter, SVCT2. The α2α3-dKO brain showed significantly decreased level of ASC compared with the wild-type (WT) and single knockout. We found that the ASC content in the basal ganglia and cerebellum was significantly lower in the adult Atp1a3 heterozygous knockout mouse (α3-HT) than in the WT. Interestingly, we observed a significant decrease in the ASC level in the basal ganglia and cerebellum of α3-HT in the peripartum period, during which mice are under physiological stress. These observations indicate that the α2 and α3 subunits independently contribute to the ASC level in the foetal brain and that the α3 subunit contributes to ASC transport in the adult basal ganglia and cerebellum. We propose that decreases in ASC levels may affect neural network development and are linked to the pathophysiology of ATP1A2- and ATP1A3-related neurologic disorders.

Comparison of Intravenous and Periarticular Administration of Corticosteroids in Total Knee Arthroplasty: A Prospective, Randomized Controlled Study.

BACKGROUND: Corticosteroids are widely used in total knee arthroplasty (TKA) to relieve postoperative pain and prevent postoperative nausea. The aim of this prospective, randomized controlled study was to compare the effects of intravenous and periarticular administration of corticosteroids on pain control, prevention of postoperative nausea, and inflammation and thromboembolism markers following TKA. METHODS: One hundred patients undergoing TKA were randomly allocated to either the intravenous administration or periarticular injection group. The intravenous administration group received 10 mg dexamethasone 1 hour before and 24 hours after the surgical procedure, as well as a periarticular injection placebo during the procedure. The periarticular injection group received a 40-mg injection of triamcinolone acetonide during the surgical procedure, as well as an intravenous administration placebo 1 hour before and 24 hours after the procedure. Postoperative pain scores at rest and during walking and nausea scores were recorded according to the 0-to-10 Numerical Rating Scale. Interleukin-6 (IL-6), C-reactive protein (CRP), and prothrombin fragment 1.2 (PF1.2) were measured preoperatively and postoperatively. RESULTS: Pain scores at rest and during walking 24 hours postoperatively were significantly lower in the periarticular injection group than in the intravenous administration group. Nausea scores showed no significant difference between groups. IL-6 at 24 and 48 hours postoperatively also showed no significant difference between groups. CRP at 24 and 48 hours postoperatively was significantly lower in the intravenous administration group than in the periarticular injection group. In contrast, CRP at 1 week postoperatively was significantly higher in the intravenous administration group than in the periarticular injection group. The mean PF1.2 was significantly lower in the intravenous administration group than in the periarticular injection group at 4 hours postoperatively. Two cases of deep venous thrombosis in each group were detected with use of ultrasonographic examination. CONCLUSIONS: Periarticular injection of corticosteroids showed a better pain-control effect at 24 hours postoperatively than did intravenous administration, whereas the antiemetic effect was similar between treatments. Although intravenous administration had a better anti-thromboembolic effect than periarticular injection, the incidence of deep venous thrombosis was low in both groups. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Optogenetic Approach to Local Neuron Network Analysis of the Medullary Respiratory Center.

Using an optogenetic approach, we analyzed a local neuron network of the respiratory center in the medulla of a brainstem-spinal cord preparation isolated from neonatal rat. We developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch) or one of the step-function channelrhodopsin variants (ChRFR) under the control of Phox2b promoter-enhancer regions. Then, in en bloc preparations from 0- to 2-day-old Tg neonatal rats, we analyzed membrane potential changes of medullary respiratory-related neurons in response to photostimulation of the rostral ventral medulla. The photostimulation-induced inhibition or facilitation of the respiratory rhythm in Arch-expressing or ChRFR-expressing Tg rat preparations, respectively. Selective photoactivation of Phox2b-positive neurons expressing ChRFR in the rostral ventrolateral medulla of a neonatal rat en bloc preparation induced membrane potential changes of respiratory-related neurons that were dependent on heterogeneous properties of synaptic connections in the respiratory center. We concluded that the optogenetic approach is a powerful method of verifying a hypothetical model of local networks among respiratory-related neurons in the rostral ventrolateral medulla of neonatal rat.

Characteristics of cortical spreading depression and c-Fos expression in transgenic mice having a mutation associated with familial hemiplegic migraine 2.

BACKGROUND: Cortical spreading depression is thought to be the underlying mechanism of migraine aura. In 2006, three relatives having the point mutation E700K in ATP1A2 exon 15 were diagnosed with familial hemiplegic migraine 2 characterized by complicated forms of aura. Here, we generated a transgenic mouse model having the human E700K mutation in the Atp1a2 orthologous gene. OBJECTIVE: To investigate the characteristics of cortical spreading depression in a mouse model with E700K mutation in the Atp1a2. METHODS: Cortical spreading depression was induced by applying stepwise increases of KCl concentration or electrical stimulation intensity to C57BL/6J-Tg(Atp1a2*E700K)9151Kwk mice (Tg, both sexes) and corresponding wild-type animals. Under urethane anesthesia, the responsiveness and threshold to cortical spreading depression were examined and the distribution of c-Fos expression, a neuronal activity marker, was immunohistochemically determined. RESULTS: Overall, Tg mice showed significantly faster propagation velocity (p < 0.01) and longer full-width-at-half-maximum (p < 0.01) than wild-type animals, representing a slower recovery from direct current potential deflection. The cortical spreading depression threshold tended to be lower in Tg, especially in females. c-Fos-positive cells were significantly enhanced in the ipsilateral somatosensory cortex, piriform cortex, amygdala and striatum (each p < 0.05 vs. contralateral side). Numbers of c-Fos positive cells were significantly higher in the ipsilateral amygdala of Tg, as compared with wild-type animals (p < 0.01). CONCLUSION: The effect of cortical spreading depression may be greater in E700K transgenic mice than that in wild-type animals, while the threshold for cortical spreading depression shows little change. Higher c-Fos expression in the amygdala may indicate alterations of the limbic system in Tg, suggesting an enhanced linkage between cortical spreading depression and amygdala connectivity in familial hemiplegic migraine 2 patients.

Long-Term Safety and Efficacy of Sitagliptin for Type 2 Diabetes Mellitus in Japan: Results of a Multicentre, Open-Label, Observational Post-Marketing Surveillance Study.

INTRODUCTION: A post-marketing surveillance (PMS) study was conducted to confirm the long-term risk-benefit profile of sitagliptin administered to Japanese patients with type 2 diabetes mellitus (T2DM) under real-world conditions. METHODS: This prospective, multicentre, open-label PMS collected data from 3326 patients receiving sitagliptin according to the approved indication during the case registration period (July 2010-June 2012; observation period, 3 years). Safety was assessed via collection of data on adverse drug reactions (ADRs), estimated glomerular filtration rate (eGFR) and cardiovascular events whereas efficacy was assessed via changes in glycated hemoglobin (HbA1c). RESULTS: In 3265 patients evaluated for safety, 270 ADRs occurred in 207 (6.3%) patients overall. Metabolism and nutrition disorders were the most common class of ADRs, occurring in 58 patients overall (53 non-serious, 5 serious) with hypoglycaemia (17 patients, 0.52%) the most common ADR. In patients with eGFR > 90 mL/min/1.73 m2 at baseline (mean ± SD, 106.42 ± 18.11 mL/min/1.73 m2, n = 584), eGFR declined by 11.83 ± 17.53 mL/min/1.73 m2 (P < 0.0001; n = 360) over the observation period whereas eGFR appeared to be relatively maintained in patients with lower baseline eGFR levels. Cardiovascular events were infrequent [occurring in 4 of 84 (4.76%) patients at high cardiovascular risk] with no distinct features in this Japanese population and the cumulative incidence [8.42% (3.12-21.70) at 36 months; n = 32] was similar to that noted in previous studies involving sitagliptin. In patients evaluated for efficacy, the overall change in HbA1c from baseline to final evaluation was mean ± SD - 0.68 ± 1.34% (P < 0.0001, n = 2070). Reductions in HbA1c tended to be greater in younger patients and patients with higher body mass index (BMI) and HbA1c values at the start of administration. CONCLUSION: Long-term sitagliptin administration in the routine clinical practice setting is associated with good efficacy, including as monotherapy, with no additional safety concerns.

Six1 is required for signaling center formation and labial-lingual asymmetry in developing lower incisors.

BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown. RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos. CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme.

Antiviral and Virucidal Activities of Umesu Phenolics on Influenza Viruses.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.