Immunolocalization Of Protein Research Articles

Immunohistochemical Localization of Proteins in the Phloem: Problems and Solutions.

Immunolocalization of proteins in differentiated phloem cells is a challenging task given their special anatomy, organellar infrastructure, and the phloem tissue's heterogeneity. Incorporation of specific wall components in the thickened cell walls of phloem cells is often the source of unspecific labeling, leading to erroneous localization. Therefore, special care is required regarding generation and purification of specific antibodies. In addition, tissue preservation of phloem cells, which contain a high osmotic pressure in their functional state, is a very challenging task prone to various pitfalls. This chapter provides practical advice for cautious tissue preparation and antibody purification. Furthermore, methods that can be used to verify immunohistochemical localization data, such as promoter-reporter studies or activity tests, are discussed. Such confirmation experiments are essential for unambiguous determination of protein location in cells of the phloem.

Different approaches for assessing sperm function

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction.In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways.Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their sub-population distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.

Immuno-localization of ESCRT Proteins in Virus-Infected Cells by Fluorescence and Electron Microscopy.

Many enveloped viruses utilize the cellular ESCRT pathway for budding, even flaviviruses, which form viral particles inside replication organelles derived from the endoplasmic reticulum (ER). In this section, we introduce methods for detecting several ESCRT subunit proteins in virus-infected cells by immunofluorescence microscopy and immunoelectron microscopy (immuno-EM). We also introduce a new method; correlative light microscopy and electron microscopy (CLEM), which allows the observation of target structures with both high-resolution EM and fluorescence labeling.

Localizing Genome Segments and Protein Products of a Multipartite Virus in Host Plant Cells

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. This concept applied to multipartite viruses-which have a genome composed of two or more nucleic acid segments, each individually encapsulated-implies that all segments constituting a viral genome need to coinfect the same host cell for replication to occur. Would this requirement be verified, it would constitute a major cost for extreme cases of multipartition such as the Faba bean necrotic stunt virus (FBNSV, nanovirus) whose genome is composed of eight complementary segments, each encoding a single gene ( Grigoras et al., 2009 ). To address this question, we followed the distribution of the FBNSV genome segments by fluorescence in situ hybridization combined to immunolocalization of the replication-controlling viral protein within the cells of the host plant: Vicia Faba. A rapid and efficient protocol to localize viral transcripts in plant and insect hosts has been developed earlier ( Ghanim et al., 2009 ). We here improve this method by using random-primed labeled probes and apply it to the detection and quantification of the individual segments composing the FBNSV genome. Moreover, we combine this technique with immunolocalization so that both viral segments and proteins can be visualized within the same samples.

Иммунолокализация липид-транспортирующих белков в растениях гороха в норме и при засолении

Иммунолокализация липид-транспортирующих белков в растениях гороха в норме и при засолении

Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes.

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation. However, the analysis of meiotic spindle structure by immunofluorescence is of outstanding importance in plant reproductive biology and is very challenging. In the following report, we describe the complete and easy protocol for the localization of proteins to the male meiotic spindle and male meiotic chromosomes. The protocol is fast, improved, and robust without the use of any harsh enzymes.

Immunolocalization of VirB11 protein in the Anaplasma marginale outer membrane and its reaction with bovine immune sera

Type IV secretion system is composed by a group of proteins associated with substrate transfer between bacteria and eukaryotic organisms. VirB11 is a protein of this system and, in A. marginale it induces partial protection in cattle immunized with external membrane fragments of the rickettsia. The objectives of this study were: to determine the conservation rate of VirB11 among different Mexican isolates and to corroborate its cellular localization on the outer membrane of A. marginale. Nucleotide and amino acid sequence alignment of virB11 from nine Mexican isolates showed 100 % identity. Bovine sera from both naturally and experimentally infected cattle reacted against a multiple antigenic peptide (MAP‑VirB11) designed based on a B-type epitope present in VirB11. In addition, MAP‑VirB11‑rabbit immune sera reacted with native VirB11 by ELISA and western blot in rickettsial extracts and on the surface of bovine infected erythrocytes by indirect immunofluorescence and the localization of VirB11 protein was determined on the outer surface of A. marginale initial bodies.

Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar, Lepisosteus oculatus, an actinopterygian bony fish

ABSTRACTEnameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Pre-and postnatal exposition to fluorides induce changes in rats liver morphology by impairment of antioxidant defense mechanisms and COX induction

IntroductionFluorides are common in the environment and are absorbed mostly in the stomach and gut, it can easily move through cell membranes and its accumulation can cause harmful effects in skeletal and soft tissues. One of the most important F- accumulation sites is the liver.The aim of this study was to determine whether F- can cause inflammation in rat liver by affecting the activity of antioxidant enzymes and changes in the synthesis of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2). Materials and methodsAn in vivo model of prenatal and postnatal exposure to sodium fluoride (NaF) was used to carry out the experiment. Animals from control group received tap water to drink, while animals exposed to F- received drinking water containing NaF, 50 mg/L. In serum and liver we analyzed F- concentration, in liver - antioxidant enzymes activity, PGE2 and TXB2 concentration and immunolocalization of COX1 and COX2 proteins were measured. ResultsWe observed significant changes in F- concentration only in liver. The results of this study showed that F- affects antioxidant enzymes activity, COX2 protein expression and PGE2 synthesis in liver. Also, in some regions of the liver of rats exposed to F-, the hepatocytes were diffusely altered, with changes resembling microvesicular steatosis. ConclusionChronic exposure to F− during development causes an accumulation of this element in the liver and changes in antioxidant enzymes activity and cyclooxygenase expression. Long term exposure to this element is toxic to the liver and can cause disturbances in its homeostasis.

Immune response in goats vaccinated with thiol-binding proteins from Haemonchus contortus.

The experimental protocol of immunization tested here confirms its protective effect against Haemonchus contortus in goats. This protection translated into a 65.5% mean reduction in adult worm burden after a homologous challenge, and a significant decrease (73.2%) in cumulative faecal egg counts (FECs). These parasitological findings were consistent with the levels of some biopathological parameters. Thus, the reduction in adult worms and FEC observed in immunized animals were associated with increased levels of packed cell volume as well as plasma proteins. This response seems to be related to an important increase in specific antibodies (in serum and gastric mucus) and eosinophilia in response to challenge. At the local level, a cellular response was also observed in which CD4+ lymphocytes and globule leucocytes played a predominant role. Finally, it should be noted that the study of immunolocalization of proteins used in the vaccination trial suggests that these antigens have an internal location (at intestinal and reproductive tissues) in the adult worm. This observation, in conjunction with the kinetics of specific antibody levels after the challenge, suggests that these antigens may be part of excretory/secretory (E/S) products.

Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation

Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signaling cues were found to display inherent similarities and differences of phenotype. Investigation of transcription factors implicated in driving urothelial-type differentiation revealed buccal epithelial cells to have minimal or absent expression of PPARG, GATA3 and FOXA1 genes. Retroviral overexpression of protein coding sequences for GATA3 or PPARy1 in buccal epithelial cells resulted in nuclear immunolocalisation of the respective proteins, with both transductions also inducing expression of the urothelial differentiation-associated claudin 3 tight junction protein. PPARG1 overexpression alone entrained expression of nuclear FOXA1 and GATA3 proteins, providing objective evidence of its upstream positioning in a transcription factor network and identifying it as a candidate factor for urothelial-type transdifferentiation or reprogramming.

Combined effects of resveratrol and epigallocatechin-3-gallate on post thaw boar sperm and IVF parameters

Frozen-thawed boar semen suffer a fertility decrease that negatively affects its widespread use. In recent years supplementing frozen-thawed boar sperm with different antioxidants gave interesting and promising results; the aim of the present work was to study the effect of supplementing boar sperm thawing medium for 1 h with combination of epigallocatechin-3-gallate (EGCG, 50 μM) and Resveratrol (R, 2 mM), on boar sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function, lipid peroxidation and DNA integrity (assessed by flow cytometry), protein tyrosine phosphorylation (assessed by immunofluorescence) and on in vitro fertilization (IVF).Our results demonstrate that sperm motility is negatively affected by R (alone or associated with EGCG, p < 0.05) in comparison to control and EGCG groups both at 1 h and 4 h; this effect is evident both in average motility parameters and in single cells kinematics, studied by cluster analysis, that showed the presence of a specific cell population with simil-hyperactivated features in R group (p < 0.01).Viability, acrosome integrity, mitochondrial functionality and lipid peroxidation are not influenced by the addition of the antioxidants; finally, DNA integrity is negatively influenced by R (both alone or associated with EGCG) both at 1 h and 4 h incubation (p < 0.05). Finally, tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, is not affected by the different treatments.Penetration rate is strongly enhanced by R, both alone or associated with EGCG (p < 0.05); EGCG increases penetration rate as well but to a lower extent.Our findings demonstrate that the combination of R and EGCG could positively affect frozen-thawed boar sperm fertility in vitro; the effect is evident also in R groups, thus demonstrating that this antioxidant is predominant, and no synergic effect is present. Some insights are needed to understand if, in particular R (that showed the strongest effect) could be profitably used for artificial insemination in vivo, given the detrimental effect of this molecule on both sperm motility and DNA integrity.

Group I chitin deacetylases are essential for higher order organization of chitin fibers in beetle cuticle

Roles in the organization of the cuticle (exoskeleton) of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi. Even though all TcCDAs are co-expressed in cuticle-forming cells from the hardened forewing (elytron) and ventral abdomen, as well as in the softer hindwing and dorsal abdomen, there are significant differences in the tissue specificity of expression of the alternatively spliced transcripts. Loss of either TcCDA1 or TcCDA2 protein by RNAi causes abnormalities in organization of chitinous horizontal laminae and vertical pore canals in all regions of the procuticle of both the hard and soft cuticles. Simultaneous RNAi for TcCDA1 and TcCDA2 produces the most serious abnormalities. RNAi of either TcCDA2a or TcCDA2b affects cuticle integrity to some extent. Following RNAi, there is accumulation of smaller disorganized fibers in both the horizontal laminae and pore canals, indicating that TcCDAs play a critical role in elongation/organization of smaller nanofibers into longer fibers, which is essential for structural integrity of both hard/thick and soft/thin cuticles. Immunolocalization of TcCDA1 and TcCDA2 proteins and effects of RNAi on their accumulation indicate that these two proteins function in concert exclusively in the assembly zone in a step involving the higher order organization of the procuticle.

Human Placental Stem Cell Therapy in Stroke: Endothelial/Smooth Muscle Mechanisms Underlying Protection

Following thrombolytic therapy in stroke, ischemia/reperfusion (I/R) mediated inflammation impairs maintenance of blood brain barrier (BBB), enhance expression of endothelial and leukocyte adhesion molecules and perturbs normal blood flow regulation. Currently, benefits of stem cell therapy (SCT) in stroke are thought to reflect secretion of protective and trophic factors which limit acute brain I/R injury. We studied human placenta mesenchymal stem cells (hPMSC) as a potent, high‐yield and inexpensive tool in SCT for human stroke therapy. We found that acute intravenous hPMSC administration significantly reduced infarct size (p&lt;0.05) at 24h in our acute (1h) model of middle cerebral artery occlusion (MCAO) in rats. Our hypothesis was that acute benefits of hPMSCs in stroke might reflect suppression of post‐I/R immune, inflammatory and BBB disturbances which limit the onset and progression of stroke injury following restoration of blood flow. We first examined hPMSC support of BBB via mobilization of junctional proteins in human brain endothelial cells (hCMEC‐D3) in conjunction with FITC‐Dextran and FITC‐biotin‐gelatin avidin permeability assays plus western blotting and immunolocalization of junctional proteins. We also assessed the ability of hPMSC to suppress inflammation driven hCMEC‐D3 binding of macrophages (RAW246.7). Lastly we studied the influence of hPMSC on contractility of human brain vascular smooth muscle cells (hBVSMC) in collagen gel contraction assays.hPMSC significantly (p&lt;0.001) enhanced barrier integrity at 24h in oxygen/glucose deprived/reperfused (OGDR) brain endothelial monolayers (in both FITC‐dextran and FITC‐biotin assays. We found that hCMEC‐D3 which were contact‐‐independently co‐cultured with hPMSCs expressed significantly higher levels of VE‐cadherin and α‐catenin under OGDR conditions (p&lt;0.001); surprisingly, we did not see significant increases in occludin or claudin‐5 expression. Additionally, the spatial localization of VE‐cadherin and α‐catenin at hCMEC‐D3 junctions was apparently increased under OGDR, but not control conditions after hPMSC treatment. We also saw that hPMSC significantly (p&lt;0.0001) reduced binding of macrophages to activated brain endothelial monolayers. Lastly, we found that hBVSMC contraction was significantly increased by hPMSC (p=0.0001), an effect which was reversed by Angiotensin II Type 1 receptor inhibition (Losartan, 10 uM) (p=0.004), but not by thromboxane A2 or endothelin‐1 blockade.We conclude that hPMSC release stable factors which beneficially and acutely influence endothelial barrier integrity, interactions with immune cells and vasoregulation to improve recovery in stroke.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional Characteristics, Electrophysiological and Antennal Immunolocalization of General Odorant-Binding Protein 2 in Tea Geometrid, Ectropis obliqua.

As one of the main lepidopteran pests in Chinese tea plantations, Ectropis obliqua Warren (tea geometrids) can severely decrease yields of tea products. The olfactory system of the adult tea geometrid plays a significant role in seeking behaviors, influencing their search for food, mating partners, and even spawning grounds. In this study, a general odorant-binding protein (OBP) gene, EoblGOBP2, was identified in the antennae of E. obliqua using reverse transcription quantification PCR (RT-qPCR). Results showed that EoblGOBP2 was more highly expressed in the antennae of males than in females relative to other tissues. The recombinant EoblGOBP2 protein was prepared in Escherichia coli and then purified through affinity chromatography. Ligand-binding assays showed that EoblGOBP2 had a strong binding affinity for some carbonyl-containing tea leaf volatiles (e.g., (E)-2-hexenal, methyl salicylate, and acetophenone). Electrophysiological tests confirmed that the male moths were more sensitive to these candidate tea plant volatiles than the female moths. Immunolocalization results indicated that EoblGOBP2 was regionally confined to the sensilla trichoid type-II in the male antennae. These results indicate that EoblGOBP2 may be primarily involved in the olfactory activity of male E. obliqua moths, influencing their ability to sense tea leaf volatiles. This study provides a new perspective of insect GOBPs and implies that olfactory function can be used to prevent and control the tea geometrid.

A cryotechnique-based method for low abundance protein immunolocalization in tomato (Solanum lycopersicum) roots infected with a nematode, Globodera rostochiensis

Plant-parasitic cyst forming nematodes induce in host roots a specific feeding site called a syncytium. Modifications induced by the pathogen in cells incorporated into syncytium include their hypertrophy and changes in apoplast caused by over-expression of plant proteins, e.g. cellulases. As a result cell wall openings between syncytial elements are formed. The major aim of our investigation was to immunolocalize cellulases involved in these cell-wall modifications. Experiments were conducted on tomato (Solanum lycopersicum cv. “Money Maker”) infected with Globodera rostochiensis. Root segments containing syncytia were processed using two techniques: conventional method of embedding in LR-White resin and cryotechnique of progressive lowering of temperature (PLT). It is believed that the latter is superior to other techniques in keeping in place cell components and preserving antigenicity of macromolecules. It is especially useful when low abundance proteins have to be immunodetected at their place of action. The main principle of the PLT technique is a stepwise lowering of temperature throughout probe dehydration, infiltration and embedding in an appropriate resin. Two-step immunolocalization and visualization using fluorochrome (FITC) at light microscopy level or colloidal gold particles at transmission electron microscopy level was performed in this study. The labeling of cellulase 7 protein at both microscopy levels was more intensive and specific on PLT-treated sections as compared to sections obtained from the classical method. Our results confirm the usefulness of the PLT cryotechnique for plant immunocytochemistry and indicate that in nematode-infected roots cellulase 7 is predominantly present in the syncytia.

Effect of levofloxacin, pazufloxacin, enrofloxacin, and meloxicam on the immunolocalization of ABCG-2 transporter protein in rabbit retina.

Adenosine triphosphate-binding cassette (ABC) sub-family G member-2 (ABCG-2) is a transporter protein, implicated for multi-drug efflux from tissues. This study evaluated the effect of fluoroquinolones; levofloxacin, pazufloxacin and enrofloxacin, and non-steroidal anti-inflammatory drug, meloxicam; on the immunolocalization of ABCG-2 transporter protein of rabbit retinas. Thirty-two male rabbits were randomly divided in to eight groups. Control group was gavaged, 2% benzyl alcohol in 5% dextrose since these chemicals are excipients of the drug preparations used in the treatment groups of this study. Four groups were exclusively gavaged, levofloxacin hemihydrate (10mg/kg body weight b.i.d 12h), pazufloxacin mesylate (10mg/kg body weight b.i.d 12h), enrofloxacin (20mg/kg body weight o.d.), and meloxicam (0.2mg/kg body weight o.d.), respectively. Three other groups were co-gavaged meloxicam with above fluoroquinolones, respectively. These drugs were administered for 21days. ABCG-2 immunolocalization was mild in the retinas of control and levofloxacin-alone-treated groups. The immunolocalization intensity was significantly higher in meloxicam-alone-treated group when compared to control and levofloxacin-alone-treated groups. Immunolocalization of this transporter increased in the levofloxacin-meloxicam co-treated group when compared to the levofloxacin-alone-treated group. Highest immunolocalization was observed in the enrofloxacin-meloxicam co-treated group although the immunolocalization of all treatment groups, except the levofloxacin-alone-treated group, was significantly higher than the control and levofloxacin-alone-treated groups.

Inhibition of Dopamine Receptor in Neonate Hippocampus: Immunolocalization of Post Synaptic Density Protein-95 and Dopamine Receptor in vivo

The effect of haloperidol on neonatal dopaminergic neurotransmission in the hippocampus of postnatal day 20 rats (P.20) was investigated in this study. Haloperidol blocked dopamine receptors (D2R) and inhibited D2R on the membrane of neonate neurons. For this study the 0.5 ml (20 mg/kg) of haloperidol was administered to pregnant female animals intraperitoneally a week before delivery. At day P.20, 5 control animals and 5 haloperidol treated animals were taken to the behavioral studies room for the Y maze and Novel object recognition test, which was done 7 am in the morning before mating. Electrophysiology was done with 2 control pups and 2 treated pups. Electrodes were implanted in the brain at the hippocampal region 2 mm beneath the bregma, 2 mm lateral to the midline. Anterior Posterior (AP=0), Medial Lateral (ML=2 mm). Also immunolocalization and immunofluorescence of post synaptic density protein (PSD-95), hippocampal morphology and hippocampal neurons have been done respectively. Results from this study showed a decline in memory index for the Y maze as a result of the effect of D2R blockade thereby inhibiting neurotransmission in newborns. Electrophysiology result in this study showed an increase in the root mean square (RMS) of control pups. The increase in RMS is equivalent to increase in wave burst pattern caused by neuronal excitation. Immunochemistry result showed an increase in the number of PSD-95 in the hippocampus of an increase in tyrosine hydroxylase in the hippocampus of the treated neonatal rats when compared to the control neonatal rats Immunofluorescence showed decline in the number of neurons in the haloperidol treated rats and it also caused hippocampal damage in terms of morphology. Furthermore, results from electrophysiology showed a statistical significant difference with P value 0.04229 (P<0.05) using the student t-test. These findings suggest that D2R inhibition may cause decline in memory function, impair learning in newborns and disrupt neonatal dopaminergic neurotransmission.

Transient Receptor Potential Channel, Vanilloid 5, Induces Chondrocyte Apoptosis in a Rat Osteoarthritis Model Through the Mediation of Ca2+ Influx

Background/Aims: Chondrocyte apoptosis is the most common pathological feature in cartilage in osteoarthritis (OA). Transient receptor potential channel vanilloid 5 (TRPV5) is important in regulating calcium ion (Ca²⁺) influx. Accumulating evidences suggest that Ca²⁺ is a major intracellular second messenger that can trigger cell apoptosis. Therefore, we investigate the potential role of TRPV5 in mediating Ca²⁺ influx to promote chondrocyte apoptosis in OA. Methods: The monoiodoacetic acid (MIA)-induced rat OA model was assessed by macroscopic and radiographic analyses. Calmodulin protein immunolocalization was detected by immunohistochemistry. The mRNA and protein level of TRPV5, calmodulin and cleaved caspase-8 in articular cartilage were assessed by real time polymerase chain reaction and western blotting. Primary chondrocytes were isolated and cultured in vitro. TRPV5 small interfering RNA was used to silence TRPV5 in chondrocytes. Then, calmodulin and cleaved caspase-8 were immunolocalized by immunofluorescence in chondrocyte. Fluo-4AM staining was used to assess intracellular Ca²⁺ to reflect TRPV5 function of mediation Ca²⁺ influx. Annexin V-fluorescein isothiocyanatepropidium iodide flow cytometric analysis was performed to determine chondrocytes apoptosis. Western blotting techniques were used to measure the apoptosis-related proteins in chondrocyte level. Results: Here, we reported TRPV5 was up-regulated in MIA-induced OA articular cartilage. Ruthenium red (a TRPV5 inhibitor) can relieve progression of joint destruction in vivo which promoted us to demonstrate the effect of TRPV5 in OA. We found that TRPV5 had a specific role in mediating extracellular Ca²⁺ influx leading to chondrocytes apoptosis in vitro. The apoptotic effect was inhibited even reversed by silencing TRPV5. Furthermore, we found that the increase Ca²⁺ influx triggered apoptosis by up-regulating the protein of death-associated protein, FAS-associated death domain, cleaved caspase-8, cleaved caspase-3, cleaved caspase-6, and cleaved caspase-7, and the up-regulated proteins were abolished by silencing TRPV5 or 1, 2-bis-(o-Aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (a Ca²⁺ chelating agent). Conclusion: The up-regulated TRPV5 could used be as an initiating factor that induces extrinsic chondrocyte apoptosis via the mediation of Ca²⁺ influx. These findings suggested TRPV5 could be an intriguing mediator for drug target in OA.

Modulation of heat shock protein immunolocalization in cerebral cortex by melatonin therapy in heat stressed rats

Abstract Heat stress leads clinically to manifestations of central nervous system dysfunction. Heat shock proteins (HSPs) function as molecular chaperones protecting the cells from damage. Melatonin has therapeutic and prophylactic