Effect of fluid replacement with green tea on body fluid balance and renal responses under mild thermal hypohydration: a randomized crossover study.

PURPOSE: Maintaining an appropriate hydration level by ingesting fluid in a hot environment is a measure to prevent heat-related illness. Caffeine-containing beverages, including green tea (GT), have been avoided as inappropriate rehydration beverages to prevent heat-related illness because caffeine has been assumed to exert diuretic/natriuretic action. However, the influence of caffeine intake on urine output in dehydrated individuals is not well documented. The aim of the present study was to examine the effect of fluid replacement with GT on body fluid balance and renal water and electrolyte handling in mildly dehydrated individuals. METHODS: Subjects were dehydrated by performing three bouts of stepping exercise for 20 min separated by 10 min of rest. They were asked to ingest an amount of water (H2O), GT, or caffeinated H2O (20 mg/100 ml; Caf-H2O) that was equal to the volume of fluid loss during the dehydration protocol; fluid balance was measured for 2 h after fluid ingestion. RESULTS: The dehydration protocol induced hypohydration by ~ 10 g/kg body weight (~ 1% of body weight). Fluid balance 2 h after fluid ingestion was significantly less negative in all trials, and the fluid retention ratio was 52.2 ± 4.2% with H2O, 51.0 ± 5.0% with GT, and 47.9 ± 6.2% with Caf-H2O; those values did not differ among the trials. After rehydration, urine output, urine osmolality, and urinary excretions of osmotically active substances, sodium, potassium and chloride were not different among the trials. CONCLUSION: The data indicate that ingestion of GT or an equivalent caffeine amount does not worsen the hydration level 2 h after ingestion and can be effective in reducing the negative fluid balance for acute recovery from mild hypohydration. TRIAL REGISTRATION: ISRCTN53057185; retrospectively registered.

Phosphorylated CRMP1, axon guidance protein, is a component of spheroids and is involved in axonal pathology in amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS), neurodegeneration is characterized by distal axonopathy that begins at the distal axons, including the neuromuscular junctions, and progresses proximally in a "dying back" manner prior to the degeneration of cell bodies. However, the molecular mechanism for distal axonopathy in ALS has not been fully addressed. Semaphorin 3A (Sema3A), a repulsive axon guidance molecule that phosphorylates collapsin response mediator proteins (CRMPs), is known to be highly expressed in Schwann cells near distal axons in a mouse model of ALS. To clarify the involvement of Sema3A-CRMP signaling in the axonal pathogenesis of ALS, we investigated the expression of phosphorylated CRMP1 (pCRMP1) in the spinal cords of 35 patients with sporadic ALS and seven disease controls. In ALS patients, we found that pCRMP1 accumulated in the proximal axons and co-localized with phosphorylated neurofilaments (pNFs), which are a major protein constituent of spheroids. Interestingly, the pCRMP1:pNF ratio of the fluorescence signal in spheroid immunostaining was inversely correlated with disease duration in 18 evaluable ALS patients, indicating that the accumulation of pCRMP1 may precede that of pNFs in spheroids or promote ALS progression. In addition, overexpression of a phospho-mimicking CRMP1 mutant inhibited axonal outgrowth in Neuro2A cells. Taken together, these results indicate that pCRMP1 may be involved in the pathogenesis of axonopathy in ALS, leading to spheroid formation through the proximal progression of axonopathy.

Somatic cell and innate immune responses in mammary glands of lactating cows to intramammary infusion of Bifidobacterium breve at pre-drying off period.

Intramammary infusion of Bifidobacterium breve (B. breve)-induced somatic cell (SC) counts, chemiluminescent response (CL), lactoferrin (LF) concentrations and mastitis-causing pathogens from quarters with subclinical mastitis were measured to evaluate innate immune response of mammary glands in dairy cows at 3 to 4 weeks before drying off. SC counts in 7 quarters of 7 control cows and 5 quarters of 6 cows with mastitis increased markedly on day 1 and SC values in control cows were significantly (P<0.05) increased and returned to pre-infusion levels on day 5 after B. breve-infusion. CL values in both groups increased markedly on day 1 and then decreased after B. breve-infusion; however, CL values in cows with mastitis did not return to normal levels on day 5 and at postpartum. The CL values were highly correlated with their SC counts in milk from both groups. LF concentrations increased toward day 3 after B. breve-infusion and were higher in cows with mastitis. B. breve-infusion eliminated 16.6% (1/6) of pathogens from 6 quarters with chronic subclinical mastitis. B. breve-induced SC responses in quarters from 3 cows with mastitis showed characteristic patterns of recovery, persistent and new infections. B. breve-induced SC counts in quarters from the cows in the pre-drying off were lower (25.7-70.6%) than those of the cows in mid-lactation. The intrinsic innate immune response in cows on pre-drying off may be decreased and appears to be insufficient to eliminate pathogens from mammary gland in the pre-drying off.

Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia.

There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2's activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients <40 y old, we observed that nonphosphorylated CRMP2 was higher than in controls, while phosphorylated CRMP2 remained unchanged from control. In the brain, these changes were associated with dendritic structural abnormalities. The abundance of active CRMP2 with insufficient opposing inactive p-CRMP2 yielded a unique lowering of the p-CRMP2:CRMP2 ratio in SCZ patients, implying a disruption in the normal equilibrium between active and inactive CRMP2. These clinical data suggest that measuring CRMP2 and p-CRMP2 in peripheral blood might reflect intracerebral processes and suggest a rapid, minimally invasive, sensitive, and specific adjunctive diagnostic aid for early SCZ: increased CRMP2 or a decreased p-CRMP2:CRMP2 ratio may help cinch the diagnosis in a newly presenting young patient suspected of SCZ (versus such mimics as mania in bipolar disorder, where the ratio is high).

Activity-induced secretion of semaphorin 3A mediates learning.

The semaphorin family is a well-characterized family of secreted or membrane-bound proteins that are involved in activity-independent neurodevelopmental processes, such as axon guidance, cell migration, and immune functions. Although semaphorins have recently been demonstrated to regulate activity-dependent synaptic scaling, their roles in Hebbian synaptic plasticity as well as learning and memory remain poorly understood. Here, using a rodent model, we found that an inhibitory avoidance task, a hippocampus-dependent contextual learning paradigm, increased secretion of semaphorin 3A in the hippocampus. Furthermore, the secreted semaphorin 3A in the hippocampus mediated contextual memory formation likely by driving AMPA receptors into hippocampal synapses via the neuropilin1-plexin A4-semaphorin receptor complex. This signaling process involves alteration of the phosphorylation status of collapsin response mediator protein 2, which has been characterized as a downstream molecule in semaphorin signaling. These findings implicate semaphorin family as a regulator of Hebbian synaptic plasticity and learning.

Phosphorylation of Collapsin Response Mediator Protein 1 (CRMP1) at Tyrosine 504 residue regulates Semaphorin 3A-induced cortical dendritic growth.

Collapsin response mediator proteins (CRMPs) have been identified as mediating proteins of repulsive axon guidance cue Semaphorin-3A (Sema3A). Phosphorylation of CRMPs plays a crucial role in the Sema3A signaling cascade. It has been shown that Fyn phosphorylates CRMP1 at Tyrosine 504 residue (Tyr504); however, the physiological role of this phosphorylation has not been examined. We found that CRMP1 was the most strongly phosphorylated by Fyn among the five members of CRMPs. We confirmed Tyr504 phosphorylation of CRMP1 by Fyn. Immunocytochemistry of mouse dorsal root ganglion (DRG) neurons showed that phosphotyrosine signal in the growth cones was transiently increased in the growth cones upon Sema3A stimulation. Tyr504-phosphorylated CRMP1 also tended to increase after Sema3A simulation. Ectopic expression of a single amino acid mutant of CRMP1 replacing Tyr504 with phenylalanine (CRMP1-Tyr504Phe) suppressed Sema3A-induced growth cone collapse response in chick DRG neurons. CRMP1-Tyr504Phe expression in mouse hippocampal neurons also suppressed Sema3A but not Sema3F-induced growth cone collapse response. Immunohistochemistry showed that Tyr504-phosphorylated CRMP1 was present in the cell bodies and in the dendritic processes of mouse cortical neurons. CRMP1-Tyr504Phe suppressed Sema3A-induced dendritic growth of primary cultured mouse cortical neurons as well as the dendritic development of cortical pyramidal neurons in vivo. Fyn± ; Crmp1± double heterozygous mutant mice exhibited poor development of cortical layer V basal dendrites, which was the similar phenotype observed in Sema3a-/- , Fyn-/- , and Crmp1-/- mice. These findings demonstrate that Tyr504 phosphorylation of CRMP1 by Fyn is an essential step of Sema3A-regulated dendritic development of cortical pyramidal neurons. (247 words).

Effects of luminance contrast and character size on reading speed.

Both luminance contrast and character size are critical factors affecting reading performance. Previous studies reported on the effect of luminance contrast on the reading-speed function, that is, the relationship between reading speed and character size. In particular, when contrast was reduced, the critical print size (CPS) was found to shift to a larger character size even though the maximum reading speed and function shape did not change [Japanese Journal of Ophthalmology 52(1) (2008) 44-47]. In the present study, the effect of luminance contrast on the reading function was quantitatively examined. Japanese phrases with a luminance contrast of 0.03-0.99 were prepared as stimuli. Observers with normal vision were asked to read aloud phrases with several character sizes. Then, the reading functions were obtained for each luminance contrast. CPS was found to increase as the luminance contrast decreased. The relationship between contrast and CPS was linear in log-log coordinates, that is, log-CPS increased as the log-contrast of the characters decreased. It was found that the contrast of the stimulus systematically affects the location of the reading function.

Characterization of the gastric motility response to human motilin and erythromycin in human motilin receptor-expressing transgenic mice.

Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin. The distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red. hMTLR expression was abundant in the gastric smooth muscle layer. Interestingly, higher levels of hMTLR expression were observed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice. The contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca2+-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice. Human motilin and erythromycin stimulate gastric smooth muscle contraction in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca2+. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents.

CRMP2-binding compound, edonerpic maleate, accelerates motor function recovery from brain damage.

Brain damage such as stroke is a devastating neurological condition that may severely compromise patient quality of life. No effective medication-mediated intervention to accelerate rehabilitation has been established. We found that a small compound, edonerpic maleate, facilitated experience-driven synaptic glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic-acid) receptor delivery and resulted in the acceleration of motor function recovery after motor cortex cryoinjury in mice in a training-dependent manner through cortical reorganization. Edonerpic bound to collapsin-response-mediator-protein 2 (CRMP2) and failed to augment recovery in CRMP2-deficient mice. Edonerpic maleate enhanced motor function recovery from internal capsule hemorrhage in nonhuman primates. Thus, edonerpic maleate, a neural plasticity enhancer, could be a clinically potent small compound with which to accelerate rehabilitation after brain damage.

Laboratory demonstration of a cryogenic deformable mirror for wavefront correction of space-borne infrared telescopes.

This paper demonstrates a cryogenic deformable mirror (DM) with 1020 actuators based on micro-electrical mechanical systems (MEMS) technology. Cryogenic space-borne infrared telescopes can experience a wavefront error due to a figure error of their mirror surface, which makes the imaging performance worse. For on-orbit wavefront correction as one solution, we developed a MEMS-processed electro-static DM with a special surrounding structure for use under the cryogenic temperature. We conducted a laboratory demonstration of its operation in three cooling cycles between 5 K and 295 K. Using a laser interferometer, we detected the deformation corresponding to the applied voltages under the cryogenic temperature for the first time. The relationship between voltages and displacements was qualitatively expressed by the quadratic function, which is assumed based on the principle of electro-static DMs. We also found that it had a high operating repeatability of a few nm root-mean-square and no significant hysteresis. Using the measured values of repeatability, we simulated the improvement of the point spread function (PSF) by wavefront correction with our DM. These results show that our developed DM is effective in improving imaging performance and PSF contrast of space-borne infrared telescopes.

Comprehensive behavioral study and proteomic analyses of CRMP2-deficient mice.

Collapsin response mediator protein 2 (CRMP2) plays a key role in axon guidance, dendritic morphogenesis and cell polarization. CRMP2 is implicated in various neurological and psychiatric disorders. However, in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2-/- ) mice and examined their behavioral phenotypes. During 24-h home cage monitoring, the activity level during the dark phase of crmp2-/- mice was significantly higher than that of wild-type (WT) mice. Moreover, the time during the open arm of an elevated plus maze was longer for crmp2-/- mice than for WT mice. The duration of social interaction was shorter for crmp2-/- mice than for WT mice. Crmp2-/- mice also showed mild impaired contextual learning. We then examined the methamphetamine-induced behavioral change of crmp2-/- mice. Crmp2-/- mice showed increased methamphetamine-induced ambulatory activity and serotonin release. Crmp2-/- mice also showed altered expression of proteins involved in GABAergic synapse, glutamatergic synapse and neurotrophin signaling pathways. In addition, SNAP25, RAB18, FABP5, ARF5 and LDHA, which are related genes to schizophrenia and methamphetamine sensitization, are also decreased in crmp2-/- mice. Our study implies that dysregulation of CRMP2 may be involved in pathophysiology of neuropsychiatric disorders.

CRMP1 and CRMP2 have synergistic but distinct roles in dendritic development.

Collapsin response mediator protein 2, CRMP2, has been identified as an intracellular signaling mediator for Semaphorin 3A (Sema3A). CRMP2 plays a key role in axon guidance, dendritic morphogenesis, and cell polarization. It has been also implicated in a variety of neurological and psychiatric disorders. However, the in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene-deficient (crmp2(-/-) ) mice. The crmp2(-/-) mice showed irregular development of dendritic spines in cortical neurons. The density of dendritic spines was reduced in the cortical layer V pyramidal neurons of crmp2(-/-) mice as well as in those of sema3A(-/-) and crmp1(-/-) mice. However, no abnormality was found in dendritic patterning in crmp2(-/-) compared to wild-type (WT) neurons. The level of CRMP1 was increased in crmp2(-/-) , but the level of CRMP2 was not altered in crmp1(-/-) compared to WT cortical brain lysates. Dendritic spine density and branching were reduced in double-heterozygous sema3A(+/-) ;crmp2(+/-) and sema3A(+/-) ;crmp1(+/-) mice. The phenotypic defects had no genetic interaction between crmp1 and crmp2. These findings suggest that both CRMP1 and CRMP2 mediate Sema3A signaling to regulate dendritic spine maturation and patterning, but through overlapping and distinct signaling pathways.

Voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic development.

Growing axons rely on local signaling at the growth cone for guidance cues. Semaphorin3A (Sema3A), a secreted repulsive axon guidance molecule, regulates synapse maturation and dendritic branching. We previously showed that local Sema3A signaling in the growth cones elicits retrograde retrograde signaling via PlexinA4 (PlexA4), one component of the Sema3A receptor, thereby regulating dendritic localization of AMPA receptor GluA2 and proper dendritic development. In present study, we found that nimodipine (voltage-gated L-type Ca(2+) channel blocker) and tetrodotoxin (TTX; voltage-gated Na(+) channel blocker) suppress Sema3A-induced dendritic localization of GluA2 and dendritic branch formation in cultured hippocampal neurons. The local application of nimodipine or TTX to distal axons suppresses retrograde transport of Venus-Sema3A that has been exogenously applied to the distal axons. Sema3A facilitates axonal transport of PlexA4, which is also suppressed in neurons treated with either TTX or nimodipine. These data suggest that voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic GluA2 localization and branch formation.

Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A.

Mice lacking collapsin response mediator protein 1 manifest hyperactivity, impaired learning and memory, and impaired prepulse inhibition.

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that are involved in various aspects of neuronal development such as axonal guidance and neuronal migration. Here we provide evidence that crmp1 (-/-) mice exhibited behavioral abnormalities related to schizophrenia. The crmp1 (-/-) mice exhibited hyperactivity and/or impaired emotional behavioral phenotype. These mice also exhibited impaired context-dependent memory and long-term memory retention. Furthermore, crmp1 (-/-) mice exhibited decreased prepulse inhibition, and this phenotype was rescued by administration of chlorpromazine, a typical antipsychotic drug. In addition, in vivo microdialysis revealed that the methamphetamine-induced release of dopamine in prefrontal cortex was exaggerated in crmp1 (-/-) mice, suggesting that enhanced mesocortical dopaminergic transmission contributes to their hyperactivity phenotype. These observations suggest that impairment of CRMP1 function may be involved in the pathogenesis of schizophrenia. We propose that crmp1 (-/-) mouse may model endophenotypes present in this neuropsychiatric disorder.

Serotonin mediates cross-modal reorganization of cortical circuits.

Loss of one type of sensory input can cause improved functionality of other sensory systems. Whereas this form of plasticity, cross-modal plasticity, is well established, the molecular and cellular mechanisms underlying it are still unclear. Here, we show that visual deprivation (VD) increases extracellular serotonin in the juvenile rat barrel cortex. This increase in serotonin levels facilitates synaptic strengthening at layer 4 to layer 2/3 synapses within the barrel cortex. Upon VD, whisker experience leads to trafficking of the AMPA-type glutamate receptors (AMPARs) into these synapses through the activation of ERK and increased phosphorylation of AMPAR subunit GluR1 at the juvenile age when natural whisker experience no longer induces synaptic GluR1 delivery. VD thereby leads to sharpening of the functional whisker-barrel map at layer 2/3. Thus, sensory deprivation of one modality leads to serotonin release in remaining modalities, facilitates GluR1-dependent synaptic strengthening, and refines cortical organization.