The calcium-binding activity of fish scale protein hydrolysates

The calcium-binding activity of tilapia scale protein hydrolysates sequentially hydrolyzed by trypsin, flavor enzyme and pepsin were investigated. The hydrolysates were divided into four fractions using G-15 gel chromatography, and the F3 fraction has the higher calcium-binding activity of 196.3 mg/g. The UV-vis and the Fourier transform infrared spectroscopy (FTIR) demonstrate that the amino nitrogen atoms and the oxygen atoms belonging to the carboxylate groups are the primary binding sites for Ca2+. The X-ray diffraction and scanning electron microscopy (SEM) confirmed the reaction between the peptde and calcium. The results obtained indicated that this fish scale protein hydroly-sates have potential as functional foods for calcium-supplementation.

A novel method for evaluating microglial activation using ionized calcium-binding adaptor protein-1 staining:cell body to cell size ratio

Aim:The aim was to validate a newly developed methodology of semi-automatic image analysis to analyze microglial morphology as marker for microglial activation in ionized calcium-binding adaptor protein-1(IBA-1)stained brain sections.Methods:The novel method was compared to currently used analysis methods,visual characterization of activation stage and optical density measurement,in brain sections of young and aged rats that had undergone surgery or remained naïve.Results:The cell body to cell size ratio of microglia was strongly correlated to the visual characterization activation stage.In addition,we observed specific surgery and age-related changes in cell body size,size of the dendritic processes and cell body to cell size ratio.Conclusion:The novel analysis method provides a sensitive marker for microglial activation in the rat brain,which is quick and easy to perform and provides additional information about microglial morphology.

Exercise combined with administration of adipose-derived stem cells ameliorates neuropathic pain after spinal cord injury

Experimental studies have shown that exercise and human adipose-derived stem cells(ADSCs)play positive roles in spinal cord injury(SCI).However,whether ADSCs and/or exercise have a positive effect on SCI-induced neuropathic pain is still unclear.Thus,there is a need to explore the effects of exercise combined with administration of ADSCs on neuropathic pain after SCI.In this study,a thoracic 11(T11)SCI contusion model was established in adult C57BL/6 mice.Exercise was initiated from 7 days post-injury and continued to 28 days post-injury,and approximately 1×105 ADSCs were transplanted into the T11 spinal cord lesion site immediately after SCI.Motor function and neuropathic pain-related behaviors were assessed weekly using the Basso Mouse Scale,von Frey filament test,Hargreaves method,and cold plate test.Histological studies(Eriochrome cyanine staining and immunohistochemistry)were performed at the end of the experiment(28 days post-injury).Exercise combined with administration of ADSCs partially improved early motor function(7,14,and 21 days postinjury),mechanical allodynia,mechanical hypoalgesia,thermal hyperalgesia,and thermal hypoalgesia.Administration of ADSCs reduced white and gray matter loss at the lesion site.In addition,fewer microglia and astrocytes(as identified by expression of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein,respectively)were present in the lumbar dorsal horn in the SCI+ADSCs and SCI+exercise+ADSCs groups compared with the sham group.Our findings suggest that exercise combined with administration of ADSCs is beneficial for the early recovery of motor function and could partially ameliorate SCIinduced neuropathic pain.

Effect of acetyl-L-carnitine on hypersensitivity in acute recurrent caerulein-induced pancreatitis and microglial activation along the brain’s pain circuitry

BACKGROUND Acute pancreatitis(AP)and recurring AP are serious health care problems causing excruciating pain and potentially lethal outcomes due to sepsis.The validated caerulein-(CAE)induced mouse model of acute/recurring AP produces secondary persistent hypersensitivity and anxiety-like behavioral changes for study.AIM To determine efficacy of acetyl-L-carnitine(ALC)to reduce pain-related behaviors and brain microglial activation along the pain circuitry in CAE-pancreatitis.METHODS Pancreatitis was induced with 6 hly intraperitoneal(i.p.)injections of CAE(50μg/kg),3 d a week for 6 wk in male C57BL/6J mice.Starting in week 4,mice received either vehicle or ALC until experiment’s end.Mechanical hypersensitivity was assessed with von Frey filaments.Heat hypersensitivity was determined with the hotplate test.Anxiety-like behavior was tested in week 6 using elevated plus maze and open field tests.Microglial activation in brain was quantified histologically by immunostaining for ionized calcium-binding adaptor molecule 1(Iba1).RESULTS Mice with CAE-induced pancreatitis had significantly reduced mechanical withdrawal thresholds and heat response latencies,indicating ongoing pain.Treatment with ALC attenuated inflammation-induced hypersensitivity,but hypersensitivity due to abdominal wall injury caused by repeated intraperitoneal injections persisted.Animals with pancreatitis displayed spontaneous anxiety-like behavior in the elevated plus maze compared to controls.Treatment with ALC resulted in increased numbers of rearing activity events,but time spent in“safety”was not changed.After all the abdominal injections,pancreata were translucent if excised at experiment’s end and opaque if excised on the subsequent day,indicative of spontaneous healing.Post mortem histopathological analysis performed on pancreas sections stained with Sirius Red and Fast Green identified wide-spread fibrosis and acinar cell atrophy in sections from mice with CAE-induced pancreatitis that was not rescued by treatment with ALC.Microglial Iba1 immunostaining was significantly increased in hippocampus,thalamus(intralaminar nuclei),hypothalamus,and amygdala of mice with CAE-induced pancreatitis compared to naïve controls but unchanged in the primary somatosensory cortex compared to naïves.CONCLUSION CAE-induced pancreatitis caused increased pain-related behaviors,pancreatic fibrosis,and brain microglial changes.ALC alleviated CAE-induced mechanical and heat hypersensitivity but not abdominal wall injury-induced hypersensitivity caused by the repeated injections.

Effects of epinephrine on angiogenesis-related gene expressions in cultured rat cardiomyocytes

Epinephrine is often used for the treatment of patients with heart failure,low cardiac output and cardiac arrest.It can acutely improve hemodynamic parameters;however,it does not seem to improve longer term clinical outcomes.Therefore,we hypothesized that epinephrine may induce unfavorable changes in gene expression of cardiomyocyte.Thus,we investigated effects of epinephrine exposure on the mediation or modulation of gene expression of cultured cardiomyocytes at a genome-wide scale.Our investigation revealed that exposure of cardiomyocytes to epinephrine in an in vitro environment can up-regulate the expression of angiopoietin-2 gene(+2.1 times),and down-regulate the gene expression of neuregulin 1(-3.7 times),plasminogen activator inhibitor-1(-2.4 times) and SPARC-related modular calcium-binding protein-2(-4.5 times).These changes suggest that epinephrine exposure may induce inhibition of angiogenesis-related gene expressions in cultured rat cardiomyocytes.The precise clinical significance of these changes in gene expression,which was induced by epinephrine exposure,warrants further experimental and clinical investigations.

DISTRIBUTION OF PARVALBUMIN, CALBINDIN-D28 AND CALRETININ IMMUNOREACTIVE NEURONS AND FIBERS IN THE MONKEY BASAL GANGLIA

Objective To investigate the cellular localization of parvalbumin (PV), calbindin-D28k (CB) and calretinin (CR) in the monkey basal ganglia.Methods Immunocytochemical technique was used to detect PV,CB and CR immunoreactivity in the basal ganglia. Results In the striatum, CB labeled medium-sized spiny projection neurons whereas PV and CR marked two separate classes of aspiny interneurons. The striatal matrix compartment was markedly enriched with CB while striatal patches displayed a CR-rich neuropil. In the pallidum, virtually all neurons contained PV but none express CB. CR occured only in a small subpopulation of large and small pallidal neurons. In the subthalamic nucleus, there existed a multitude of PV-positive cells and fibers but the number of CR and CB-positive neuronal elements was small. In the substantia nigra / ventral tegmental area complex, CB and CR occured principally in dopaminergic neurons of the dorsal tier of the pars compacta and in those of the ventral tegmental area. PV was strickly confined to the GABAergic neurons of the pars reticular and lateralis. CB-rich fibers abounded in the pars reticular and lateralis, while CR-positive axons were confined to the pars compacta. Conclusion CB and PV were distributed according to a strikingly complementary pattern in primate basal ganglia, and the use of CB and PV immunocytochemistry may be considered as an excellent tool to define distinct chemoarchitectonic and functional domains within the complex organization of the basal ganglia. CR was less ubiquitous but occured in small basal ganglia components where it labeled distinct subsets of neurons. Such highly specific patterns of distribution indicate that CB, PV and CR may work in synery within primate basal ganglia.

Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance plasma membrane H^(+)-ATPase activity and alkali stress tolerance by inhibiting PP2C-mediated dephosphorylation of TaHA2 in wheat

Alkali stress is a major constraint for crop production in many regions of saline-alkali land.However,little is known about the mechanisms through which wheat responds to alkali stress.In this study,we identified a calcium ion-binding protein from wheat,TaCCD1,which is critical for regulating the plasma membrane(PM)H^(+)-ATPase-mediated alkali stress response.PM H+-ATPase activity is closely related to alkali tolerance in the wheat variety Shanrong 4(SR4).We found that two D-clade type 2C protein phosphatases,TaPP2C.D1 and TaPP2C.D8(TaPP2C.D1/8),negatively modulate alkali stress tolerance by dephosphorylating the penultimate threonine residue(Thr926)of TaHA2 and thereby inhibiting PM H+-ATPase activity.Alkali stress induces the expression of TaCCD1 in SR4,and TaCCD1 interacts with TaSAUR215,an early auxin-responsive protein.These responses are both dependent on calcium signaling triggered by alkali stress.TaCCD1 enhances the inhibitory effect of TaSAUR215 on TaPP2C.D1/8 activity,thereby promoting the activity of the PM H^(+)-ATPase TaHA2 and alkali stress tolerance in wheat.Functional and genetic analyses verified the effects of these genes in response to alkali stress,indicating that TaPP2C.D1/8 function downstream of TaSAUR215 and TaCCD1.Collectively,this study uncovers a new signaling pathway that regulates wheat responses to alkali stress,in which Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance PM H+-ATPase activity and alkali stress tolerance by inhibiting TaPP2C.D1/8-mediated dephosphorylation of PM H+-ATPase TaHA2 in wheat.

S100A8 promotes epithelial-mesenchymal transition and metastasis under TGF-β/USF2 axis in colorectal cancer

Background:The transforming growth factor-β(TGF-β)pathway plays a pivotal role in inducing epithelial-mesenchymal transition(EMT),which is a key step in cancer invasion and metastasis.However,the regulatory mechanism of TGF-βin inducing EMT in colorectal cancer(CRC)has not been fully elucidated.In previous studies,it was found that S100A8 may regulate EMT.This study aimed to clarify the role of S100A8 in TGF-β-induced EMT and explore the underlying mechanism in CRC.Methods:S100A8 and upstream transcription factor 2(USF2)expression was detected by immunohistochemistry in 412 CRC tissues.Kaplan-Meier survival analysis was performed.In vitro,Western blot,and migration and invasion assays were performed to investigate the effects of S100A8 and USF2 on TGF-β-induced EMT.Mouse metastasis models were used to determine in vivo metastasis ability.Luciferase reporter and chromatin immunoprecipitation assay were used to explore the role of USF2 on S100A8 transcription.Results:During TGF-β-induced EMT in CRC cells,S100A8 and the transcription factor USF2 were upregulated.S100A8 promoted cell migration and invasion and EMT.USF2 transcriptionally regulated S100A8 expression by directly binding to its promoter region.Furthermore,TGF-βenhanced the USF2/S100A8 signaling axis of CRC cells whereas extracellular S100A8 inhibited the USF2/S100A8 axis of CRC cells.S100A8 expression in tumor cells was associated with poor overall survival in CRC.USF2 expression was positively related to S100A8 expression in tumor cells but negatively related to S100A8-positive stromal cells.Conclusions:TGF-βwas found to promote EMT and metastasis through the USF2/S100A8 axis in CRC while extracellular S100A8 suppressed the USF2/S100A8 axis.USF2 was identified as an important switch on the intracellular and extracellular S100A8 feedback loop.

Revealing the Precise Role of Calretinin Neurons in Epilepsy: We Are on the Way

Epilepsy is a common neurological disorder characterized by hyperexcitability in the brain.Its pathogenesis is classically associated with an imbalance of excitatory and inhibitory neurons.Calretinin(CR)is one of the three major types of calcium-binding proteins present in inhibitory GABAergic neurons.The functions of CR and its role in neural excitability are still unknown.Recent data suggest that CR neurons have diverse neurotransmitters,morphologies,distributions,and functions in different brain regions across various species.Notably,CR neurons in the hippocampus,amygdala,neocortex,and thalamus are extremely susceptible to excitotoxicity in the epileptic brain,but the causal relationship is unknown.In this review,we focus on the heterogeneous functions of CR neurons in different brain regions and their relationship with neural excitability and epilepsy.Importantly,we provide perspectives on future investigations of the role of CR neurons in epilepsy.

Structural basis for prokaryotic calciummediated regulation by a Streptomyces coelicolor calcium binding protein

The important and diverse regulatory roles of Ca2+in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily.However,the calcium-regulatory proteins in prokaryotes are still poorly understood.In this study,we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor,named CabD,which shares low sequence homology with other known helix-loop-helix EF-hand proteins.The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins.The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins,including the bending conformation of the first C-terminalα-helix,unpaired ligand-binding EF-hands and the lack of the extreme Cterminal loop region,suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes,and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.