Zeta Protein Research Articles

Inactivation of Staphylococcus aureus and Escherichia coli in milk by different processing sequences of ultrasound and heat

AbstractDifferent processing sequences of ultrasound and heat were applied for the inactivation of Staphylococcus aureus and Escherichia coli in milk. The changes in whey protein content, particle size, zeta potential, pH, viscosity, and color values were studied to evaluate the effects on the quality of milk. Results indicated ultrasound treatment (600 W, 5 min) with postheating (63 °C 5 min) reduced S. aureus and E. coli by 1.58 and 2.02 log cfu/ml, respectively, but yielded 0.26 and 0.19 log cfu/ml sublethal S. aureus and E. coli. Milk preheated to 63 °C (5 min) followed by ultrasound (600 W, 5 min) showed 1.17 and 1.68 log reductions for S. aureus and E. coli, respectively, and the sublethal S. aureus and E. coli were reduced to 0.16 and 0.09 log cfu/ml. The increased cavitation effects make the simultaneous treatment (600 W, 63 °C, 5 min) the most effective approach in killing S. aureus and E. coli, causing 1.67 and 2.16 log reductions, respectively, and producing negligible sublethal bacteria. The simultaneous treatment was also able to improve some physical–chemical properties of milk; smaller particle size and whiter color were achieved. However, whey protein and stability indices of milk were slightly deteriorated by this approach.Practical applicationsIn food processing fields, thermosonication can serve as an alternative processing technique to thermal pasteurization. Especially, the simultaneous treatment of ultrasound and heat showed best inactivation effect for viable and sublethal cells, while maintaining the overall milk quality in the meantime. The results of this research may be useful for the treatment of various bacterial contamination and give the guidance for milk processing.

Trichinella britovi muscle larvae and adult worms: stage-specific and common antigens detected by two-dimensional gel electrophoresis-based immunoblotting

BackgroundTrichinella britovi is the second most common species of Trichinella that may affect human health. As an early diagnosis of trichinellosis is crucial for effective treatment, it is important to identify sensitive, specific and common antigens of adult T. britovi worms and muscle larvae. The present study was undertaken to uncover the stage-specific and common proteins of T. britovi that may be used in specific diagnostics.MethodsSomatic extracts obtained from two developmental stages, muscle larvae (ML) and adult worms (Ad), were separated using two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. The positively-visualized protein spots specific for each stage were identified through liquid chromatography-tandem mass spectrometry (LC-LC/MS).ResultsA total of 272 spots were detected in the proteome of T. britovi adult worms (Ad) and 261 in the muscle larvae (ML). The somatic extracts from Ad and ML were specifically recognized by T. britovi-infected swine sera at 10 days post infection (dpi) and 60 dpi, with a total of 70 prominent protein spots. According to immunoblotting patterns and LC-MS/MS results, the immunogenic spots recognized by different pig T. britovi-infected sera were divided into three groups for the two developmental stages: adult stage-specific proteins, muscle larvae stage-specific proteins, and proteins common to both stages. Forty-five Ad proteins (29 Ad-specific and 16 common) and thirteen ML proteins (nine ML-specific and four common) cross-reacted with sera at 10 dpi. Many of the proteins identified in Ad (myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin) are involved in structural and motor activity. Among the most abundant proteins identified in ML were 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. Heat-shock protein, intermediate filament protein ifa-1 and intermediate filament protein B were identified in both proteomes.ConclusionsTo our knowledge, this study represents the first immunoproteomic identification of the antigenic proteins of adult worms and muscle larvae of T. britovi. Our results provide a valuable basis for the development of diagnostic methods. The identification of common components for the two developmental stages of T. britovi may be useful in the preparation of parasitic antigens in recombinant forms for diagnostic use.

Physical stability and microstructure of rapeseed protein isolate/gum Arabic stabilized emulsions at alkaline pH

The objective of this study was to investigate the effects of gum Arabic (GA) contents (0%–3%, w/v) and pH (7, 8 and 9) on the stability of 3% (w/v) rapeseed protein isolate (RPI) emulsion. The centrifugal precipitation rate, stability coefficient, interfacial adsorption protein content, droplet size and zeta potential, rheology, confocal laser scanning microscope (CLSM) and fourier transform infrared spectroscopy (FTIR) were measured. Results showed that the stability of emulsions varied with different GA contents and pH values. The most stability of RPI-stabilized emulsion was achieved when 1% of GA was added at pH 8. Under the optimal condition (1% GA being added at pH 8), the analysis of size and zeta potential of complexes revealed that the particle size was 314 nm, and largest absolute zeta potential value (−44.3 ± 0.5 mV) was obtained. CLSM microscopic observations further verified these results. In addition, the FTIR spectra showed the absorption peak of amide І and amide Ⅱ of RPI-GA complex decreased and were shifted compared to RPI alone, and the β-sheet was significantly increased whereas β-antiparallel was significantly decreased when 1% of GA was added at pH 8, indicating the interaction of RPI and GA mainly via the changing of hydrogen bonds.

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction.

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II-IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.

Methionine sulfoxide reductase A (MsrA) mediates the ubiquitination of 14-3-3 protein isotypes in brain

The methionine sulfoxide reductase (Msr) system is known for its function in reducing protein-methionine sulfoxide to methionine. Recently, we showed that one member of the Msr system, MsrA, is involved in the ubiquitination-like process in Archaea. Here, the mammalian MsrA is demonstrated to mediate the ubiquitination of the 14-3-3 zeta protein and to promote the binding of 14-3-3 proteins to alpha synuclein in brain. MsrA was also found to enhance the ubiquitination and phosphorylation of Ser129 of alpha synuclein in brain. Furthermore, we demonstrate that, similarly to the archaeal MsrA, the mammalian MsrA can compete for capturing ubiquitin using the same active site it contains for methionine sulfoxide binding. Based on our previous observations showing that MsrA knockout mice have elevated expression levels of dopamine and 14-3-3 zeta and our current data, we propose that MsrA-dependent 14-3-3 zeta ubiquitination affects the regulation of alpha synuclein degradation and dopamine synthesis in the brain.

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two–dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30–75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, β-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.

Gga-miR-451 Negatively Regulates Mycoplasma gallisepticum (HS Strain)-Induced Inflammatory Cytokine Production via Targeting YWHAZ.

Mycoplasma gallisepticum (MG) is the most economically significant mycoplasma pathogen of poultry that causes chronic respiratory disease (CRD) in chickens. Although miRNAs have been identified as a major regulator effect on inflammatory response, it is largely unclear how they regulate MG-induced inflammation. The aim of this study was to investigate the functional roles of gga-miR-451 and identify downstream targets regulated by gga-miR-451 in MG infection of chicken. We found that the expression of gga-miR-451 was significantly up-regulated during MG infection of chicken embryo fibroblast cells (DF-1) and chicken embryonic lungs. Overexpression of gga-miR-451 decreased the MG-induced inflammatory cytokine production, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), whereas inhibition of gga-miR-451 had the opposite effect. Gene expression data combined with luciferase reporter assays demonstrated that tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta (YWHAZ) was identified as a direct target of gga-miR-451 in the context of MG infection. Furthermore, upregulation of gga-miR-451 significantly inhibited the MG-infected DF-1 cells proliferation, induced cell-cycle arrest, and promoted apoptosis. Collectively, our results demonstrate that gga-miR-451 negatively regulates the MG-induced production of inflammatory cytokines via targeting YWHAZ, inhibits the cell cycle progression and cell proliferation, and promotes cell apoptosis. This study provides a better understanding of the molecular mechanisms of MG infection.

172. Receptor Protein Tyrosine Phosphatases in Schizophrenia

Receptor protein tyrosine phosphatases, gamma (PTPRZ) and zeta (PTPRG), are cell surface ligands for contactin molecules, they bind with Contactins and are crucial for cell adhesion, signaling and neurodevelopment. Their main expression is in microglia and neurons, respectively; PTPRZ is essential for recovery from neuroinflammation. Both regulate neurotransmitter receptor function and are genetically associated with schizophrenia, affective disorders, Alzheimer’s and autism.

Knockdown of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) enhances tumorigenesis both in vivo and in vitro in bladder cancer.

Bladder cancer is the most common tumor of the urinary tract. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), a gene encoding the 14-3-3ζ protein, has been observed to be frequently amplified in bladder cancer. However, the role of 14-3-3ζ in various types of cancer is controversial. With reproduction of The Cancer Genome Atlas database, we searched the correlation of YWHAZ expression with survival outcomes of multiple cancers in silico. Our results revealed that only in bladder cancer was there a positive survival trend with YWHAZ overexpression. To study its role in bladder cancer, YWHAZ was successfully downregulated by lentivirus in 5637 and T24 cells. MTT and colony-formation assays showed that YWHAZ downregulation increased cell proliferation. Wound healing and Transwell assays showed that YWHAZ downregulation enhanced cell migration and invasiveness. FACS analysis showed that YWHAZ induced cell cycle arrest, but not apoptosis. A xenograft tumor model revealed that YWHAZ knockdown enhanced tumor growth. Gene set enrichment analysis confirmed that the cell cycle pathway plays a vital role in the function of the YWHAZ gene. In conclusion, knockdown of YWHAZ promoted both in vitro and in vivo tumorigenesis in bladder cancer and may be a novel biomarker for bladder cancer deserving further study.

Beneficial effects of polysaccharides on the solubility of Mytilus edulis enzymatic hydrolysates

The low stability of proteins and peptides in liquid systems restricts the development of enzymatic hydrolysate. In this study, a variety of protein hydrolysates from Mytilus edulis (PHM) were prepared by enzymatic hydrolysis. The effects of four kinds of polysaccharides, CMC-Na, xanthan gum, β-cyclodextrin, and Z-trim, on the stability of PHM were investigated by comparing the protein precipitation rate, particle size, zeta potential, microstructure, and fluorescence spectra. Results showed that the aqueous mixture enriched with proteins and peptides was stabilized by the addition of 1 mg/mL xanthan gum at pH 8, and it significantly prevented protein deposition. Particle size and zeta potential measurements confirmed the beneficial effects of xanthan gum on the stability of PHM. This study provides some theoretical references for the research and development of proteins from M. edulis.

Identification of tarsal-less peptides from the silkworm Bombyx mori.

The polycistronic and non-canonical gene tarsal-less (tal, known as pri) was reported to be required for embryonic and imaginal development in Drosophila; however, there are few reports of the tal gene in the silkworm Bombyx mori. Here, we cloned a tal-like (Bmtal) gene, and a sequence analysis showed that the Bmtal cDNA (1661bp) contains five small open reading frames (smORFs) (A1, A2, A3, A4, and B) that encode short peptides of 11-12 (A1-A4) amino acid residues containing an LDPTG(E)L(Q)(V)Y motif that is conserved in Drosophila Tal, as well as a 32-amino-acid B peptide. Reverse transcription-quantitative polymerase chain reaction showed that the expression of the Bmtal gene was highest in the trachea, followed by the silk gland and Malpighian tubule, in day 3 fifth-instar larvae. Subcellular localization showed that BmTal localized in the nucleus. By regulating the expression of the full-length Bmtal gene and the functional smORFs of Bmtal, we showed that the expression levels of the Bmovo gene and genes related to the Notch, transforming growth factor-β, and Hippo signaling pathways changed with changes in BmTal peptide expression. A co-immunoprecipitation assay showed that BmTal interacts with polyubiquitin, which triggered degradation and/or processing of the 14-3-3 protein zeta. A comparative transcriptome analysis showed that 2843 (2045) genes were up- (down)-regulated after Bmtal gene expression was up-regulated. The up- (down)-regulated differentially expressed genes were enriched in 326 (278) gene ontology terms (P ≤ 0.05) and 54 (59) Kyoto Encyclopedia of Genes and Genomes pathways (P ≤ 0.05), and the results indicated that the BmTal peptides could function as mediators of hormone levels or the activities of multiple pathways, including the peroxisome proliferator-activated receptor, Hedgehog, mitogen-activated protein kinase, adipocytokine, and gonadotropin-releasing hormone signaling pathways, as well as the innate immune response. These results increase our understanding of the function and mechanism of BmTal at the genome-wide level.

Inhibition of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) Overcomes Drug Resistance and Tumorigenicity in Ovarian Cancer

Background/Aims: Cancer stem-like cells are the main cause of tumor occurrence, progression, and therapeutic resistance. However, the precise signals required for the maintenance of the stem-like traits of these cells in ovarian cancer remain elusive. We have thus worked to elucidate the functional role of Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), a gene encoding the 14-3-3ζ protein, in the regulation of multidrug resistance and stem cell-like traits in ovarian cancer. Methods: We detected the YWHAZ levels in human ovarian cancer specimens and cell lines using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blots. MTS assays, soft agar colony formation assays, migration assays, cell cycle analysis, sphere formation assays, and flow cytometry were applied to investigate the functional role of YWHAZ in ovarian cancer. Results: Our data reveals substantially increased YWHAZ expression in both cisplatin- and paclitaxel-resistant ovarian cancer cells. Silencing YWHAZ restored the sensitivity of resistant ovarian cancer cells to cisplatin and paclitaxel. Furthermore, in vitro studies showed that down-regulation of YWHAZ inhibited cell cycle progression, migration, and the expression of stem cell markers. Moreover, tumorigenicity was suppressed in tumor-bearing BALB/c nude mice following YWHAZ knockdown. Additionally, we demonstrated that the expression of YWHAZ was directly down-regulated by miR-30e in resistant ovarian cancer cells. Conclusion: Our results have led to new insights into the essential role of YWHAZ in the regulation of tumourigenesis, stem-like traits, and drug resistance in ovarian cancer, thereby helping to identify a potential target for ovarian cancer therapy.

Reference gene selection for gene expression study in shell gland and spleen of laying hens challenged with infectious bronchitis virus

Ten reference genes were investigated for normalisation of candidate target gene expression data in the shell gland and spleen of laying hens challenged with two strains of infectious bronchitis virus (IBV). Data were analysed with geNorm, NormFinder and BestKeeper, and a comprehensive ranking (geomean) was calculated. In the combined data set of IBV challenged shell gland samples, the comprehensive ranking showed TATA-box binding protein (TBP) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) as the two most stable, and succinate dehydrogenase complex flavoprotein subunit A (SDHA) and albumin (ALB) as the two least stable reference genes. In the spleen, and in the combined data set of the shell gland and spleen, the two most stable and the two least stable reference genes were TBP and YWHAZ, and ribosomal protein L4 (RPL4) and ALB, respectively. Different ranking has been due to different algorithms. Validation studies showed that the use of the two most stable reference genes produced accurate and more robust gene expression data. The two most and least stable reference genes obtained in the study, were further used for candidate target gene expression data normalisation of the shell gland and spleen under an IBV infection model.

Lure-and-Kill Yeast Interfering RNA Larvicides Targeting Neural Genes in the Human Disease Vector Mosquito Aedes aegypti

New mosquito control strategies are vitally needed to address established arthropod-borne infectious diseases such as dengue and yellow fever and emerging diseases such as Zika and chikungunya, all of which are transmitted by the disease vector mosquito Aedes aegypti. In this investigation, Saccharomyces cerevisiae (baker’s yeast) was engineered to produce short hairpin RNAs (shRNAs) corresponding to the Aedes aegypti orthologs of fasciculation and elongation protein zeta 2 (fez2) and leukocyte receptor cluster (lrc) member, two genes identified in a recent screen for A. aegypti larval lethal genes. Feeding A. aegypti with the engineered yeasts resulted in silenced target gene expression, disrupted neural development, and highly significant larval mortality. Larvicidal activities were retained following heat inactivation and drying of the yeast into tabular formulations that induced >95% mortality and were found to attract adult females to oviposit. These ready-to-use inactivated yeast interfering RNA tablets may one day facilitate the seamless integration of this new class of lure-and-kill species-specific biorational mosquito larvicides into integrated mosquito control programs.

Involvement of miR-451 in resistance to paclitaxel by regulating YWHAZ in breast cancer

MicroRNAs (miRNAs) have been identified as major post-transcriptional regulators of the initiation and progression of human cancers, including breast cancer. However, the detail role of miR-451 has not been fully elucidated in breast cancer. In this study, we aimed to investigate the biological role and molecular mechanisms of miR-451 in drug resistance in breast cancer cell lines and in xenograft model. We show that miR-451 is decreased in human breast cancer specimens and in paclitaxel-resistant (PR) cells. Ectopic expression of miR-451 could inhibit the cell migration and invasion, promoted apoptosis, induced cell-cycle arrest Furthermore, tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein zeta (YWHAZ) was identified as a direct target of miR-451. Remarkably, the expression of YWHAZ is inversely correlated with the level of miR-451 in human breast cancer samples. Co-treatment with miR-451 mimics and YWHAZ-siRNA significantly enhanced YWHAZ knockdown in both SKBR3/PR and MCF-7/PR cells Moreover, miR-451 markedly inhibited expression of β-catenin via YWHAZ and subsequently inhibited downstream gene cyclin D1, c-Myc expression. The results of xenograft model in vivo showed that intratumor injection of miR-451 agomir induced a tumor-suppressive effect in SKBR3/PR drug-resistant xenograft model. Taken together, our findings suggested that miR-451 might be considered as important and potential target in paclitaxel-resistant breast cancer treatment.

Parallel habitat acclimatization is realized by the expression of different genes in two closely related salamander species (genus Salamandra).

The utilization of similar habitats by different species provides an ideal opportunity to identify genes underlying adaptation and acclimatization. Here, we analysed the gene expression of two closely related salamander species: Salamandra salamandra in Central Europe and Salamandra infraimmaculata in the Near East. These species inhabit similar habitat types: 'temporary ponds' and 'permanent streams' during larval development. We developed two species-specific gene expression microarrays, each targeting over 12 000 transcripts, including an overlapping subset of 8331 orthologues. Gene expression was examined for systematic differences between temporary ponds and permanent streams in larvae from both salamander species to establish gene sets and functions associated with these two habitat types. Only 20 orthologues were associated with a habitat in both species, but these orthologues did not show parallel expression patterns across species more than expected by chance. Functional annotation of a set of 106 genes with the highest effect size for a habitat suggested four putative gene function categories associated with a habitat in both species: cell proliferation, neural development, oxygen responses and muscle capacity. Among these high effect size genes was a single orthologue (14-3-3 protein zeta/YWHAZ) that was downregulated in temporary ponds in both species. The emergence of four gene function categories combined with a lack of parallel expression of orthologues (except 14-3-3 protein zeta) suggests that parallel habitat adaptation or acclimatization by larvae from S. salamandra and S. infraimmaculata to temporary ponds and permanent streams is mainly realized by different genes with a converging functionality.

Disruption to schizophrenia-associated gene Fez1 in the hippocampus of HDAC11 knockout mice

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1–9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.

Isolation and characterization of collagen extracted from channel catfish (Ictalurus punctatus) skin

Channel catfish skin is a by-product from catfish fillet production. Collagens were extracted from catfish skins by: (1) acid; (2) homogenization-aided; and (3) pepsin-aided extraction methods. Kinetic analysis of extraction was performed. SDS-PAGE was carried out for all collagens extracted under different conditions. Protein solubility, zeta potential, circular dichroism and gel strength of the collagen extracted by three methods were studied to determine optimal extraction conditions. Protein recovery rate from minced skins extracted with pH 2.4 HCl containing 23.6KU/g pepsin was the highest (64.19%). SDS-PAGE showed that collagens extracted with different methods had different proteins ratio patterns, even though the molecular mass of collagen subunits were similar, 123 and 113KDa for α1 and α2 chains, 226KDa for β chain and 338.5KDa for γ chain, respectively. Channel catfish skin collagens were typical type I collagens and could have applications in food, medical and cosmetic industries.

Expression of 14-3-3 Zeta Protein in Dexamethasone-Treated Mice and Human TM-1 Cells

ABSTRACTPurpose: 14-3-3 zeta protein plays a potential protective role in neurodegenerative disease. Given that glaucoma and neurodegenerative diseases share a similar pathogenesis, it is possible that 14-3-3 zeta may have a similar protective effect in the glaucomatous process. In the present study, we measured the expression of 14-3-3 zeta in vivo (mouse eyes) and in vitro in a transformed human trabecular meshwork (HTM) cell line, TM-1, and assessed the possible roles of this protein in dexamethasone (DEX)-treated eyes and HTM cells.Methods: Mouse eyes were randomly treated with 0.1% dexamethasone (DEX) eye drops or phosphate-buffered solution (PBS) for 28 days. The expression and distribution of 14-3-3 zeta protein in mouse eyes were examined using immunofluorescence. TM-1 cells were treated with DEX (10−6 or 10−7 M) or PBS for 1, 4, or 7 days, and the mRNA and protein expression of 14-3-3 zeta were detected by real-time RT-PCR and Western blotting.Results: 14-3-3 zeta protein was highly expressed in the mouse cornea, trabecular meshwork (TM), and ciliary body. Intraocular pressure (IOP) was significantly elevated, whereas the 14-3-3 zeta expression was significantly decreased in mouse TM after 0.1% DEX treatment for 28 days. In vitro, treatment with 10−7 M DEX mildly increased 14-3-3 zeta mRNA and protein expression (p > 0.05), whereas 10−6 M DEX significantly decreased expression of 14-3-3 zeta mRNA and protein (p < 0.05) compared to the control (Ctrl) group at the seventh day.Conclusions: DEX can increase IOP in mouse eyes and concurrently downregulate 14-3-3 zeta protein expression in mouse TM. The effects of DEX on 14-3-3 zeta expression in vitro were both dose- and time-related. Our results suggest that alterations in 14-3-3 zeta protein may be implicated in DEX-induced pathological elevated IOP.

Application of CLARITY to Investigate the 3D Architecture of 14-3-3 Zeta Protein in AD

CLARITY (Clear Lipid–exchanged Acrylamide–hybridized Rigid Imaging/ Immunostaining/ in situ–hybridization–compatible Tissue hydrogel) is a powerful, innovative, whole brain-clearing technology, and it has been successfully combined with the immunofluorescence staining to achieve the 3D visualization of some proteins or cells in mm-thick brain tissue or even the intact brains. These 3D information help to gain deeper understanding on the pathologic mechanism of some neuronal diseases (for example Parkinson and Alzheimer). 14-3-3 zeta is a highly-expressed protein in Alzheimer’s disease (AD) brain, which was closely related with the formation of Tau aggregation and neurofibrillary tangles. However, little useful information has been available concerning the 3D architecture of 14-3-3 zeta in AD disease. In this paper, the transgenic AD mice were used and the 1mm-thick brain slices were passively clarified. Immunofluorescence staining results showed that different from the control group, 14-3-3 zeta was mainly present around the hippocampus in AD mice. Additionally, the morphology of 14-3-3 zeta protein was filamentous with different lengths. This result will be helpful for exploring the in vivo role of 14-3-3 zeta protein during the progression of AD.