Biomedical subjects

Tsuyoshi Hirota

Publications and source records attributed to Tsuyoshi Hirota.

7 recordsLinked to original sources

PubMed2006

Decrease in rat internal anal pressure with the use of a topical ointment containing a killed E. coli culture suspension.

OBJECTIVES: The present study aimed to clarify the mechanisms of a topical ointment containing an Escherichia coli culture suspension and hydrocortisone (Posterisan forte, BCS+HC) in lowering internal anal pressure in conscious rats. MATERIALS AND METHODS: Internal anal pressure was measured using a water-filled balloon system for consecutive 10-min periods. The changes in pressure were evaluated by the number of peaks above 20 mmH2O between 1 and 8 min of recording. RESULTS: Topical intra-anal application of BCS+HC ointment (160 mg/kg) significantly decreased the internal anal pressure at 3 h after the application. Thereafter, this effect reached a maximum decrease at 4 h and lasted until 6 h. BCS+HC ointment (40, 80, and 160 mg/kg) lowered the internal anal pressure at 4-5 h in a dose-dependent manner. The maximum decrease ratios of the ointment and corresponding hydrocortisone-free ointment (Posterisan, BCS) were 32.6+/-12.7 and 25.7+/-9.0%, respectively, revealing significant pressure-lowering effects compared with a placebo (P<0.05). In contrast, the same ointment containing hydrocortisone alone and other ointments containing steroids or local anesthetics had no effects. DISCUSSION: Treatment with 1 mg/kg NG-nitro-L-arginine methyl ester HCl (L-NAME), a non-selective nitric oxide synthase inhibitor, significantly suppressed the effect of BCS+HC ointment (160 mg/kg) in lowering the internal anal pressure. Furthermore, BCS+HC ointment (160 mg/kg) significantly lowered capsaicin-induced high internal anal pressure compared to a placebo. CONCLUSION: These findings suggest that BCS+HC and BCS ointments containing an E. coli culture suspension significantly lowered the internal anal pressure due to endogenous nitric oxide production in conscious rats.

Administration, Topical↗

PubMed2006

Phosphorylation of mCRY2 at Ser557 in the hypothalamic suprachiasmatic nucleus of the mouse.

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.

Animals↗

PubMed2005

Ser-557-phosphorylated mCRY2 is degraded upon synergistic phosphorylation by glycogen synthase kinase-3 beta.

Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.

Active Transport, Cell Nucleus↗

PubMed2004

Resetting mechanism of central and peripheral circadian clocks in mammals.

Almost all organisms on earth exhibit diurnal rhythms in physiology and behavior under the control of autonomous time-measuring system called circadian clock. The circadian clock is generally reset by environmental time cues, such as light, in order to synchronize with the external 24-h cycles. In mammals, the core oscillator of the circadian clock is composed of transcription/translation-based negative feedback loops regulating the cyclic expression of a limited number of clock genes (such as Per, Cry, Bmal1, etc.) and hundreds of output genes in a well-concerted manner. The central clock controlling the behavioral rhythm is localized in the hypothalamic suprachiasmatic nucleus (SCN), and peripheral clocks are present in other various tissues. The phase of the central clock is amenable to ambient light signal captured by the visual rod-cone photoreceptors and non-visual melanopsin in the retina. These light signals are transmitted to the SCN through the retinohypothalamic tract, and transduced therein by mitogen-activated protein kinase and other signaling molecules to induce Per gene expression, which eventually elicits phase-dependent phase shifts of the clock. The central clock controls peripheral clocks directly and indirectly by virtue of neural, humoral, and other signals in a coordinated manner. The change in feeding time resets the peripheral clocks in a SCN-independent manner, possibly by food metabolites and body temperature rhythms. In this article, we will provide an overview of recent molecular and genetic studies on the resetting mechanism of the central and peripheral circadian clocks in mammals.

Animals↗

PubMed2003

p38 mitogen-activated protein kinase regulates oscillation of chick pineal circadian clock.

Extracellular signal-regulated kinase (ERK) and p38 are members of the mitogen-activated protein kinase (MAPK) family, and in some cases these kinases serve for closely related cellular functions within a cell. In a wide range of animal clock structures, ERK plays an important role in the circadian time-keeping mechanism. Here we found that immunoreactivity to p38 protein was uniformly distributed among cells in the chick pineal gland. On the other hand, a constant level of activated p38 was detected over the day, predominantly in the follicular and parafollicular pinealocytes that are potential circadian clock-containing cells. Chronic application of SB203580, a selective and reversible inhibitor of p38, to the cultured chick pineal cells markedly lengthened the period of the circadian rhythm of the melatonin release (up to 28.7 h). Noticeably, despite no significant temporal change of activated p38 level, a 4-h pulse treatment with SB203580 delayed the phase of the rhythm only when delivered during the subjective day. These results indicate a time-of-day-specific role of continuously activated p38 in the period length regulation of the chick pineal clock and suggest temporally separated regulation of the clock by two MAPKs, nighttime-activated ERK and daytime-working p38.

Animals↗

PubMed2002

Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts.

In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified >100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

Animals↗

PubMed2002

Prolonged anti-inflammatory action of DL-lactide/glycolide copolymer nanospheres containing betamethasone sodium phosphate for an intra-articular delivery system in antigen-induced arthritic rabbit.

PURPOSE: The objective of the present study was to develop prolonged anti-inflammatory action of DL-lactide/glycolide copolymer (PLGA) nanosphere incorporating a water-soluble corticosteroid (betamethasone sodium phosphate; BSP). Another aim was to demonstrate the biocompatibility and biologic efficacy of these BSP-loaded nanospheres directly administered into ovalbumin-induced chronic synovitis in the rabbit. METHODS: BSP-loaded nanospheres were prepared by an emulsion solvent diffusion method in oil (caprylate and caprate triglyceride). The drug releasing properties of the nanospheres were measured in vitro in phosphate buffer saline (PBS: pH7.4), and in vivo in rat air-pouch (pseudo synovial fluid). The BSP-loaded nanosphere suspensions were administered into the joint cavity in a model of antigen-induced arthritic rabbit and evaluated by measuring the joint swelling, and the biocompatibility was appraised by histologic microscopy. RESULTS: The BSP-nanospheres were a unimodally-dispersed particulate system with a mean diameter ranging from 300 to 490 nm, and BSP was efficiently entrapped in the lipophilic copolymer (PLGA), although its hydrophilic properties. The drug release-rate from the nanospheres in PBS was controlled by the molecular weight and the lactic/glycolic acid (LA/GA) ratio of the polymers. The in vitro releasing study demonstrated that sustained drug release occurred for over three weeks. In the antigen-induced arthritic rabbit, the joint swelling decreased significantly by administering BSP-loaded nanospheres during a 21-day period after intra-articular challenge. With regards to the prolonged anti-inflammatory efficacy, serum antibody to ovalbumin showed a sustained reduction during the period, and the steroidal effect appeared by the degradation of the polymer in the synovium. The BSP-nanosphere administered was phagocytosed by the synovial activated-cells and the cartilage degradation was almost prevented. CONCLUSIONS: Direct intra-articular injection of a PLGA nanosphere system with a water-soluble steroid provided a prolonged pharmacological efficacy in the joints of arthritic rabbits. The local anesthetic in the knee-joints was evaluated to be safe and without biologic damage.

Animals↗