Trajectories and Milestones of Cortical and Subcortical Development of the Marmoset Brain From Infancy to Adulthood.

With increasing attention on the developmental causes of neuropsychiatric disorders, appropriate animal models are crucial to identifying causes and assessing potential interventions. The common marmoset is an ideal model as it has sophisticated social/emotional behavior, reaching adulthood within 2 years of birth. Magnetic resonance imaging was used in an accelerated longitudinal cohort (n = 41; aged 3-27 months; scanned 2-7 times over 2 years). Splines were used to model nonlinear trajectories of grey matter volume development in 53 cortical areas and 16 subcortical nuclei. Generally, volumes increased before puberty, peaked, and declined into adulthood. We identified 3 milestones of grey matter development: I) age at peak volume; II) age at onset of volume decline; and III) age at maximum rate of volume decline. These milestones differentiated growth trajectories of primary sensory/motor cortical areas from those of association cortex but also revealed distinct trajectories between association cortices. Cluster analysis of trajectories showed that prefrontal cortex was the most heterogenous of association regions, comprising areas with distinct milestones and developmental trajectories. These results highlight the potential of high-field structural MRI to define the dynamics of primate brain development and importantly to identify when specific prefrontal circuits may be most vulnerable to environmental impact.

Information processing in brainstem bitter taste-relaying neurons defined by genetic tracing.

Bitter reception is mediated by taste receptor cells that coexpress multiple T2Rs, a family of G-protein-coupled receptors. However, it remains elusive how bitter taste information is translated in the brain into appropriate behavioral responses. Here we used a combination of genetic tracing and electrophysiological and immunohistochemical analyses in mice to functionally characterize the neurons in the solitary tract nuclei of the medulla, which receive input from mT2R5-expressing cells. The neurons defined by a transneuronal tracer originating from mT2R5-expressing cells receive glutamatergic synaptic input via the AMPA receptor. The satiety peptide cholecystokinin increases glutamatergic transmission, suggesting an interaction between information processing of taste and the homeostatic control of feeding. Nevertheless, the tracer-labeled neuron types are heterogeneous, and can be classified into catecholamine and pro-opiomelanocortin neurons. Our data reveal that the architectural solution in the first-order central relay that processes information from mT2R5-expressing cells uses unique ensembles of neurons with different neurotransmitters.

Irsogladine maleate counters the interleukin-1 beta-induced suppression in gap-junctional intercellular communication but does not affect the interleukin-1 beta-induced zonula occludens protein-1 levels in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Irsogladine maleate counters gap junctional intercellular communication reduction induced by interleukin-8 or Actinobacillus actinomycetemcomitans in cultured human gingival epithelial cells. Interleukin-1 beta is involved in periodontal disease. Little is known, however, about the effect of interleukin-1 beta on intercellular junctional complexes in human gingival epithelial cells. Furthermore, irsogladine maleate may affect the actions of interleukin-1 beta. In this study, we examined how interleukin-1 beta affected gap junctional intercellular communication, connexin 43 and zonula occludens protein-1, and how irsogladine maleate modulated the interleukin-1 beta-induced changes in the intercellular junctional complexes in human gingival epithelial cells. MATERIAL AND METHODS: Human gingival epithelial cells were exposed to interleukin-1 beta, with or without irsogladine maleate. Connexin 43 and zonula occludens protein-1 were examined at mRNA and protein levels by real-time polymerase chain reaction and western blotting, respectively. Gap junctional intercellular communication was determined using the dye transfer method. The expression of zonula occludens protein-1 was also confirmed by immunofluorescence. RESULTS: Interleukin-1 beta decreased connexin 43 mRNA levels, but increased zonula occludens protein-1 mRNA levels. Irsogladine maleate countered the interleukin-1 beta-induced reduction in gap junctional intercellular communication and connexin 43 levels. However, irsogladine maleate did not influence the increased zonula occludens protein-1 levels. CONCLUSION: The effect of interleukin-1 beta on gap junctional intercellular communication and tight junctions of human gingival epithelial cells is different. The recovery of gap junctional intercellular communication by irsogladine maleate in the gingival epithelium may be a normal process in gingival epithelial homeostasis.

Role of mastication and swallowing in the control of autonomic nervous activity for heart rate in different postures.

Mastication and swallowing increase the heart rate, and posture change and respiration also modulate the heart rate. To clarify the role of mastication and swallowing in the modulation of the autonomic nervous activity, we investigated how they interact with modulation of the heart rate by changing body positions and respiration in young healthy subjects. R-R intervals of electrocardiogram at rest were significantly changed with different body positions, compared with supine and standing. A net shortening by mastication of a chewing gum base was similar in various postures. Respiration induced a periodic change in the R-R intervals, depending on the body postures, but mastication did not markedly change them in each posture. Dry swallowing at rest and spontaneous swallowing during the mastication in the sitting position induced a similar transient shortening and suppressed the respiration-induced changes after the swallowing. The net transient shortening by dry swallowing at rest was similar in the different postures. These results suggest that signals from mastication and swallowing are summated with those from body positions and respiration for shortening the R-R intervals and that signals from swallowing suppress the respiration-induced periodic changes.

Evaluation of mastication-induced change in sympatho-vagal balance through spectral analysis of heart rate variability.

Mastication modulates the autonomic nervous activity of the digestive glands and the heart. The autonomic nervous balance is evaluated with spectral analysis of heart rate variability. In the present study, we investigated the effects of mastication of chewing gum base on heart rate variability to clarify the role of mastication in the sympatho-vagal balance for the regulation of the heart rate. Mastication of a chewing gum base stimulated the salivary secretion and shortened the R-R intervals in the electrocardiogram of healthy young subjects without swallowing of saliva at a fixed rate of respiration. Based on the analysis of heart rate variability, mastication increased the low-frequency band spectral power (LF), and decreased the high-frequency band spectral power (HF). The LF/HF was markedly increased by the mastication. Mastication enhances the sympathetic nervous activity and/or suppresses the parasympathetic nervous activity for the heart. Feeding behaviour with mastication might play a role in the modulation of the autonomic nervous activity.

Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid intralobular duct lumen: visualization of the secretory function under a confocal laser scanning microscope.

Cyclic AMP evokes fluid secretion with bicarbonate in exocrine ducts. Clearance of fluorescent dyes from rat parotid intralobular ducts by forskolin was visualized as a fluorescence change in the duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the ducts. When the isolated rat parotid intralobular duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the ducts. Methazolamide inhibited a forskolin-induced sustained decrease in duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in duct fluorescence. These findings suggest that a major portion of the steady decrease in duct fluorescence by forskolin was related to intracellular HCO3? production, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from duct space possibly due to fluid secretion in rat parotid ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

Ulnar deviation after volar subluxation of the proximal interphalangeal joint.

A rare case of irreducible and progressive ulnar deviation after volar subluxation of the proximal interphalangeal joint is presented. An immobilized proximal interphalangeal joint with remaining volar subluxation after improper reduction showed ulnar deviation at 3 weeks after injury. During surgery, the radial collateral ligament was found to be ruptured at its origin, with formation of concomitant scar tissues. There were no apparent lesions at the central slip, lateral band, and volar plate. Interposition of the ruptured ligament and infiltration of the surrounding scar tissues into the proximal interphalangeal joint were identified. Surgical incision of the capsule along the dorsal margin of the radial collateral ligament readily produced successful reduction. The irreducible and progressive ulnar deviation of the proximal interphalangeal joint seemed to result from gradual infiltration of the scar tissues, subsequent to remaining volar subluxation because of interposition of the ruptured collateral ligament.

Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains.

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.

Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.

Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3- secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 microM), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The anion current was markedly suppressed by addition of 1 mM methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3-. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3- significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl- channel located in luminal membranes) and production of cytosolic HCO3- induce the inward anion current and resulting depolarization. Inhibition of the Na(+)-K(+)-2Cl- cotransporter and the Cl(-)-HCO3- exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl- via the Na(+)-K(+)-2Cl- cotransporter or the Cl(-)-HCO3- exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3- produced intracellularly, but not of Cl- due to lack of active Cl- transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

High-level secretory production of phospholipase A1 by Saccharomyces cerevisiae and Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of lecithin to form lysolecithin. The PLA1 gene, which had been cloned from Aspergillus oryzae, was expressed in Saccharomyces cerevisiae and A. oryzae. Through the modification of the medium composition and the feeding conditions of substrate, the production level of PLA1 by S. cerevisiae was increased to a level fivefold higher than that indicated in a previous report. In the case of A. oryzae, introduction of multicopies of PLA1 expression units, and the morphological change from the pellet form to the filamentous form were effective for the enhancement of PLA1 production. We succeeded in producing 3,500 U/ml of PLA1 using an industrial-scale fermentor.

External Cl(-)-dependent formation of watery vacuoles by long-term hypotonic shock in 3T3-L1 cells.

Osmotic shock transiently induces a volume change in the cells, followed by a restoration of the cell volume due to intracellular water regulation. Effect of long-term osmotic shock on the water regulation is not completely understood. Vacuole formation by long-term osmotic shock was investigated to clarify the water exclusion mechanism from cytoplasm into intracellular vacuoles in 3T3-L1 cells. Incubation of cells in hypotonic solution reversibly induced the vacuole formation. Staining of vacuoles with fluorescent dyes revealed that vacuoles were derived from endoplasmic reticulum and Golgi apparatus but not lysosomes. Membrane-impermeable fluorescent dyes were taken up into some vacuoles from cytoplasm and extracellular solution, suggesting that some vacuoles exhibit the dynamic changes for the connection of plasma membrane, and that transporter for membrane-impermeable dyes might be active in some vacuole membranes. External Cl(-), but not Na(+), was required for vacuole formation. DPC suppressed the vacuole formation and increased cell height, and further incubation with DPC increased the number of dead cells. Bumetanide, dimethylamiloride, and HgCl(2) did not suppress the hypotonic stress-induced formation of water vacuoles. These findings suggest that 3T3-L1 cells regulate the intracellular water content through the DPC-sensitive external Cl(-)-dependent vacuole formation during long-term osmotic stress.

[A case of primary leiomyosarcoma of the pulmonary artery].

A 74-year-old woman was admitted to our hospital because of right chest pain. The chest radiograph showed right hilar pulmonary artery dilatation. A mass exhibiting low intensity in T1-weighted images and high intensity in T2-weighted images was disclosed in the right pulmonary artery. Because of its clinical course and the MRI findings, the mass was thought to be tumorous tissue, and so pneumonectomy was performed. Leiomyosarcoma was diagnosed from the histological findings. Primary artery sarcoma is rare and the prognosis is considered to be extremely poor. The patient was successfully treated and had a good clinical outcome.

Predominant role of the chorda tympani nerve in the maintenance of the taste pores: the influence of gustatory denervation in ear surgery.

The effect on the taste pores of denervation of the chorda tympani nerve in the middle-ear cavity was studied comparing confocal laser microscopy with lingual nerve resection. Taste pore cells were stained for actin with rhodamine-phalloidin and positive fluorescence was observed as a ring shape at the transverse cross sections. Within three days after chorda tympani nerve resection the ring reaction disappeared, although the pore morphology remained intact as seen by scanning electron microscopy. On the other hand, lingual nerve resection did not induce such rapid disappearance of the ring reaction. These results suggest that the chorda tympani nerve plays a predominant role in the maintenance of actin filaments in taste pore cells.

Visualization of the secretory process involved in Ca2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein.

The central feature of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholinergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein loaded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical pole. Cytoplasmic calcein was released into intercellular canaliculi just after the addition of CCh, depending upon a rise in [Ca2+]i by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles connected with intercellular canaliculi was visualized in the calcein-loaded acini, depending upon external Ca2+. Both the calcein release and vacuole formation were inhibited by suppressing the Ca(2+)-activated K+ efflux. The calcein release was also affected by the external anion substitution, suggesting that calcein is released through an anion channel. In the isolated, perfused glands, CCh-induced fluid secretion was sustained in two phases, whereas the loaded calcein was initially and transiently released into the saliva. By revealing the [Ca2+]i dependence and sensitivities to channel blockers, our results suggest that the initial phase of CCh-induced fluid secretion was evoked in association with the release of the organic anion, calcein, and the late phase of fluid secretion, during which calcein is less permeable, was associated with the formation of watery vacuoles. Thus, the anion channels possessing the distinct property of anion permeation may be activated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful for visualizing the process of Ca(2+)-dependent fluid secretion, and for clarifying the relation between fluid secretion and anion transport.

cAMP-Dependent potentiation of the Ca(2+)-activated release of the anionic fluorescent dye, calcein, from rat parotid acinar cells.

A recent study indicates that elevation of [Ca(2+)](i) enhances the release of calcein, an anionic fluorescent dye, from isolated exocrine acinar cells, so cytoplasmic calcein is useful for monitoring the secretion of organic anions. In this study, we investigated the effect of cAMP on the calcein release evoked by elevation of [Ca(2+)](i). Isoproterenol, forskolin and dibutyryl cyclic AMP (dbcAMP) did not induce the release of calcein from isolated parotid acinar cells, but they potentiated the carbachol-induced release of calcein. Although cytoplasmic calcein is released through an increase in [Ca(2+)](i), isoproterenol potentiated the carbachol-induced release of calcein without affecting the increase in [Ca(2+)](i) evoked by a high concentration of carbachol (10(-6) M). Charybdotoxin, a K(+) channel blocker, inhibited both the carbachol-induced release and the potentiation by isoproterenol. However, the calcein permeation pathways mediating the carbachol-induced release and the isoproterenol-potentiated release exhibited distinct sensitivities to anion channel blockers. Our results indicate that the calcein release induced by carbachol is potentiated through an increase in intracellular levels of cAMP. Although both the Ca(2+)-activated release and the cAMP-potentiated release may be coupled to Ca(2+)-activated K(+) efflux, increases in both [Ca(2+)](i) and [cAMP](i) may activate the calcein conduction pathway which is not activated by an increase in [Ca(2+)](i) alone.

Important role of the hepatic vagus nerve in glucose uptake and production by the liver.

We examined the role of the hepatic vagus nerve in hepatic and peripheral glucose metabolism. To assess endogenous glucose production (EGP), hepatic uptake of first-pass glucose infused intraportally (HGU), and the metabolic clearance rate of glucose (MCR), rats were subjected to hepatic vagotomy (HV, n = 7) or sham operation (SH, n = 8), after 10 days, they were then subjected to a euglycemic-hyperinsulinemic clamp together with a portal glucose load in the 24-hour fasting state. Metabolic parameters were determined by the dual-tracer method using stable isotopes. During the experiment, [6,6-2H2]glucose was continuously infused into the peripheral vein. To maintain euglycemia (4.5 mmol/L), insulin (54 pmol x kg(-1) x min(-1)) and glucose were infused peripherally after the 90-minute tracer equilibration and 30-minute basal periods, and glucose containing 5% enriched [U-13C]glucose was infused intraportally (50 micromol x kg(-1) x min(-1)) for 120 minutes (clamp period). EGP was significantly higher in HV rats versus SH rats during the basal period (64.3 +/- 7.6 v 43.6 +/- 5.3 micromol x kg(-1) x min(-1), P < .005)) and was comparable to EGP in SH rats during the clamp period (9.3 +/- 21.5 v 1.1 +/- 11.7 micromol x kg(-1) x min(-1)). HGU was reduced in HV rats compared with SH rats during portal glucose infusion (5.9 +/- 2.4 v 10.1 +/- 3.2 micromol x kg(-1) x min(-1)). The MCR in HV rats was significantly higher than in SH rats in the basal period (11.0 +/- 2.0 v 7.9 +/- 0.8 mL x kg(-1) x min(-1), P < .01)) and was comparable to the MCR in SH rats during the clamp period (41.9 +/- 10.0 and 36.6 +/- 5.7 mL x kg(-1) x min(-1)). We conclude that innervation of the hepatic vagus nerve is important for the regulation of hepatic glucose production in the postabsorptive state and HGU in the postprandial state.

Immunohistochemical observation of actin filaments in epithelial cells encircling the taste pore cavity of rat fungiform papillae.

Epithelial cells are connected to each other around taste pores in rat fungiform papillae. Cytoskeletal components are responsible for the maintenance of intracellular adhesion, and we investigated the identification and localization of actin filaments around taste pores. On the basis of observations made by immunohistochemical transmission electron microscopy comparing with confocal laser scanning microscopy using actin-lectin double staining, actin filaments were found to be localized, encircling the squeezed taste pore cavity, in epithelial cells a few micrometers below the papilla surface. In addition, these observations suggest that the organization of actin filaments around taste pores might be involved in the constriction of taste pores.

Effect of ethanol on the production of carboxypeptidase Y using the GAL10 promoter in a Saccharomyces cerevisiae gal80 mutant.

In the course of studying carboxypeptidase Y (CPY) production, we found that the expression level of the gene, which is under the control of the GAL10 promoter, increased in a Saccharomyces cerevisiae gal80 mutant grown in a medium containing ethanol as the sole carbon source. In the cultivation of the gal80 mutant KS58-2D/pCY303 carrying a multicopy plasmid, which contains the PRC1 gene fused to the GAL10 promoter, CPY production continued after the consumption of galactose. In this phase, the cells utilized ethanol as the carbon source. To increase the CPY production level, we examined the effect of carbon source feeding in a fed-batch culture. The production level in the fed-batch culture using ethanol was 1.3-fold higher than that in a batch culture and 1.6-fold higher than that in a fed-batch culture using galactose. By 5'-deletion analysis of the GAL10 promoter, the region between -256 and -232 was found to be important for the promoter activity in the gal80 mutant growing in the presence of ethanol.

Process development for industrial-scale preparation of carboxypeptidase Y secreted by Saccharomyces cerevisiae.

A method for industrial-scale preparation of carboxypeptidase Y (CPY), which was secreted by Saccharomyces cerevisiae KS58-2D/pCY303 into the culture broth, was developed. Because the purification process consists of a few simple unit operations including only one chromatography step, a higher CPY recovery was achieved than that in the process using disrupted baker's yeast. Approximately 100 g of purified CPY powder was constantly obtained using the final culture broth from a 500-l fermentor.