RESUMO
Seaweeds are important components of marine ecosystems with emerging potential in aquaculture and as sources of biofuel, food products and pharmacological compounds. However, an increasingly recognised threat to natural and industrial seaweed populations is infection with parasitic single-celled eukaryotes from the relatively understudied oomycete lineage. Here we examine the eukaryomes of diverse brown, red and green marine macroalgae collected from polar (Baffin Island), cold-temperate (Falkland Islands) and tropical (Ascension Island) locations, with a focus on oomycete and closely related diatom taxa. Using 18S rRNA gene amplicon sequencing, we show unexpected genetic and taxonomic diversity of the eukaryomes, a strong broad-brush association between eukaryome composition and geographic location, and some evidence of association between eukaryome structure and macroalgal phylogenetic relationships (phylosymbiosis). However, the oomycete fraction of the eukaryome showed disparate patterns of diversity and structure, highlighting much weaker association with geography and no evidence of phylosymbiosis. We present several novel haplotypes of the most common oomycete Eurychasma dicksonii and report for the first time a cosmopolitan distribution and absence of host specificity of this important pathogen. This indicates rich diversity in macroalgal oomycete pathogens and highlights that these pathogens may be generalist and highly adaptable to diverse environmental conditions.
Assuntos
Microbiota , Oomicetos , Filogenia , Alga Marinha , Oomicetos/genética , Oomicetos/classificação , Alga Marinha/microbiologia , Microbiota/genética , RNA Ribossômico 18S/genética , Simbiose , Biodiversidade , Eucariotos/genética , Eucariotos/classificação , Variação GenéticaRESUMO
Mechanical damage to a cell can be fatal, and the cell must reseal its membrane and restore homeostasis to survive. Plant cell repair involves additional steps such as rebuilding vacuoles, rearranging chloroplasts, and remodeling the cell wall. When we pierced a Griffithsia monilis cell with a glass needle, a large amount of intracellular contents was released, but the cell membrane resealed in less than a second. The turgor of the vacuole was quickly restored, and the punctured cell returned to its original shape within an hour. Organelles such as chloroplasts and nuclei migrated to the wound site for 12 h and then dispersed throughout the cell after the wound was covered by a new cell wall. Using fluorescent probes, high levels of reactive oxygen species (ROS) and calcium were detected at the wound site from 3 h after wounding, which disappeared when cell repair was complete. Wounding in a solution containing ROS scavengers inhibited cellular repair, and inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity or blocking calcium influx reversibly inhibited cell repair. Oryzalin reversibly inhibited both chloroplast movement and ROS production during cell repair. Our results show that cell repair in G. monilis is regulated by calcium-mediated ROS signaling and that microtubules serve as mechanical effectors.
RESUMO
Reactive oxygen species (ROS) signalling has a multitude of roles in cellular processes throughout biology. We hypothesized that red algal fertilization may offer an interesting model to study ROS-mediated signalling, as the stages of fertilization are complex and unique. We detected the localization of ROS production microscopically and monitored the expression of three homologues of NADPH oxidase in reproductive cells during fertilization. ROS were instantaneously produced by spermatia (sperm) when they attached to female trichogynes, diffused across the cell membrane in the form of H2O2, and triggered ROS generation in the carpogonium (egg) as well as carpogonial branch cells which are not in direct contact with spermatia. The expression of NADPH oxidase homologues, RESPIRATORY BURST OXIDASE HOMOLOGUES (BmRBOHs), began to be up-regulated in the female plant upon gamete binding, peaking during the fertilization process and descending back to their original level after fertilization. Pre-treatment with diphenylene iodonium or caffeine blocked gene expression as well as H2O2 production. Post-fertilization development was also inhibited when the redox state of the plants was perturbed with H2O2 at any time before or after the fertilization. Our results suggest that H2O2 acts as an auto-propagating signalling molecule, possibly through Ca2+ channel activation, and regulates gene expression in fertilization as well as post-fertilization development in red algae.
Assuntos
Peróxido de Hidrogênio , Rodófitas , Fertilização , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rodófitas/metabolismo , Transdução de SinaisRESUMO
Diverse sex determination mechanisms have been reported in eukaryotes, but little is known about the genetic pathways leading to sex determination in red algae. Sex-specific genes that could be involved in sex determination and sexual differentiation were investigated in the red alga Bostrychia moritziana by analyzing the transcriptomes of various phases including males, females, and tetrasporophytes. Sex dominantly expressed genes which showed >10-fold difference between sexes was isolated using comparative RNA-seq analysis. We found 19 gene homologues, 10 from males, and nine from females, that were found only in one sex in genomic amplification using strains collected from five different localities. Most of the sex-specific genes are involved in important cellular processes including chromosome segregation, nucleo-cytoplasmic protein shuttling, or tRNA modification. Quantitative PCR analysis showed that some sex-specific genes were differently regulated during critical events of sexual reproduction like fertilization and carposporophyte development. We could localize the expression of a male-specific gene in spermatia before and after gamete binding using RNA in situ hybridization. Amino acid sequence identity between male and female homologues of importin alpha gene and PreQ(0) reductase were highly divergent (75% and 74%, respectively), suggesting that these divergent homologues are on non-recombining UV-type chromosomes in their respective sexes. Another set of transcripts were found that were sex dominantly expressed, but not sex-specific. Nineteen out of 39 sex dominantly expressed transcripts were annotated to transposable elements. Our results suggest that sexual differentiation in B. moritziana may be achieved by multi-level regulation of cellular processes, both from genes present only in one sex and differential expression of shared genes.
Assuntos
Rodófitas , Sequência de Aminoácidos , Feminino , Perfilação da Expressão Gênica , Genoma , Masculino , Reprodução , Rodófitas/genética , TranscriptomaRESUMO
Spirogyra filaments show unique photomovement that differs in response to blue, red, and far-red light. Phototropins involved in the blue-light movement have been characterized together with downstream signaling components, but the photoreceptors and mechanical effectors of red- and far-red light movement are not yet characterized. The filaments of Spirogyra varians slowly bent and aggregated to form a tangled mass in red light. In far-red light, the filaments unbent, stretched rapidly, and separated from each other. Mannitol and/or sorbitol treatment significantly inhibited this far-red light movement suggesting that turgor pressure is the driving force of this movement. The bending and aggregating movements of filaments in red light were not affected by osmotic change. Three phytochrome homologues isolated from S. varians showed unique phylogenetic characteristics. Two canonical phytochromes, named SvPHY1 and SvPHY2, and a noncanonical phytochrome named SvPHYX2. SvPHY1 is the first PHY1 family phytochrome reported in zygnematalean algae. The gene involved in the transport of phytochromes into the nucleus was characterized, and its expression in response to red and far-red light was measured using quantitative PCR. Our results suggest that the phytochromes and the genes involved in the transport system into the nucleus are well conserved in S. varians.
Assuntos
Fitocromo , Spirogyra , Estreptófitas , Zygnematales , Luz , Filogenia , Fitocromo A , Proteínas de PlantasRESUMO
Disease outbreaks devastate Pyropia aquaculture farms every year. The three most common and serious diseases are Olpidiopsis-blight and red-rot disease caused by oomycete pathogens and green-spot disease caused by the PyroV1 virus. We hypothesized that a basic genetic profile of molecular defenses will be revealed by comparing and analyzing the genetic response of Pyropia tenera against the above three pathogens. RNAs isolated from infected thalli were hybridized onto an oligochip containing 15,115 primers designed from P. tenera expressed sequence tags (EST)s. Microarray profiles of the three diseases were compared and interpreted together with histochemical observation. Massive amounts of reactive oxygen species accumulated in P. tenera cells exposed to oomycete pathogens. Heat shock genes and serine proteases were the most highly up-regulated genes in all infection experiments. Genes involved in RNA metabolism, ribosomal proteins and antioxidant metabolism were also highly up-regulated. Genetic profiles of P. tenera in response to pathogens were most similar between the two biotrophic pathogens, Olpidiopsis pyropiae and PyroV1 virus. A group of plant resistance genes were specifically regulated against each pathogen. Our results suggested that disease response in P. tenera consists of a general constitutive defense and a genetic toolkit against specific pathogens.
Assuntos
Rodófitas , Genes de PlantasRESUMO
Lectins, characterized by their carbohydrate-binding ability, have extensive practical applications. However, their industrial use is limited due to impurity. Thus, quality-controlled production of recombinant lectin is necessary. In this study, the algal lectin BPL3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocyte. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled large-scale production of recombinant lectins.
Assuntos
Acetilgalactosamina/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Algas/metabolismo , Lectinas/metabolismo , Proteínas de Algas/química , Proteínas de Algas/isolamento & purificação , Cromatografia Líquida , Eritrócitos/metabolismo , Testes de Hemaglutinação , Humanos , Lectinas/química , Lectinas/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em TandemRESUMO
Plants and freshwater algae devoid of flagella evolved various photomovements to optimize their photosynthetic efficiency. The filaments of Spirogyra varians exhibit complex swaying and undulating movement and form a compact mat which enables them to adjust their light exposure. Photomovement of filament fragments (1-10 cells) was analyzed using various photoreceptor and cytoskeleton inhibitors under monochromatic light. Different patterns of movement were observed under red and blue light. The filaments showed positive phototropism under blue light. Under red light, the filaments bent to undulating shape, but rapidly became unbent by a short exposure to far-red light suggesting the involvement of phytochrome in this movement. The mechanical effector for the red-light response was microtubules; the movement was inhibited effectively by the microtubule inhibitor, oryzalin. The blue-light movement was partially inhibited by the single treatment of either cytochalasin D or oryzalin, but was completely blocked when both chemicals were applied together. Phototropin-signaling inhibitors, wortmannin and LY294002, reversibly inhibited the blue-light movement. Caffeine treatment reversibly stopped the blue-light movement, while the red-light movement was not affected by calcium inhibitors. Our results suggest that the complex photomovement of S. varians is the result of a two-track control of microtubules and microfilaments signaled by the combination of phytochrome and phototropin-like receptors.
Assuntos
Luz , Movimento , Spirogyra/citologia , Spirogyra/efeitos da radiação , Cálcio/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Modelos Biológicos , Spirogyra/fisiologia , Imagem com Lapso de TempoRESUMO
Mutations in the NEFH gene encoding the heavy neurofilament protein are usually associated with neuronal damage and susceptibility to amyotrophic lateral sclerosis (ALS). Recently, frameshift variants in NEFH (p.Asp1004Glnfs*58 and p.Pro1008Alafs*56) have been reported to be the underlying cause of axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC). The frameshift mutation resulted in a stop loss and translation of a cryptic amyloidogenic element (CAE) encoded by the 3' untranslated region (UTR). This study also identified a de novo c.3015_3027dup frameshift mutation predicting p.Lys1010Glnfs*57 in NEFH from a CMT2 family with an atypical clinical symptom of prominent proximal weakness. This mutation is located near the previously reported frameshift mutations, suggesting a mutational hotspot. Lower limb magnetic resonance imaging (MRI) revealed marked hyperintense signal changes in the thigh muscles compared with those in the calf muscles. Therefore, this study suggests that the stop loss and translational elongations by the 3' UTR of the NEFH mutations may be a relatively frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness.
Assuntos
Regiões 3' não Traduzidas/genética , Axônios/patologia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Mutação da Fase de Leitura/genética , Proteínas de Neurofilamentos/genética , Adulto , Doença de Charcot-Marie-Tooth/patologia , Análise Mutacional de DNA , Feminino , Humanos , Extremidade Inferior/diagnóstico por imagem , Imageamento por Ressonância Magnética , Condução Nervosa/genética , LinhagemRESUMO
Plant lectins have attracted much attention for biomedical applications including targeted drug delivery system and therapy against tumors and microbial infections. The main problem of using lectins as a biomedical tool is a batch-to-batch variation in isoforms content. The production of lectins using recombination tools has the advantage of obtaining high amounts of proteins with more precise properties, but there are only a handful of functional recombinant lectins presently available. A fetuin/asialo-fetuin specific lectin, Rhodobindin, has unique tandem repeats structure which makes it useful in exploiting for recombinant lectin. We developed three functional recombinant lectins using E. coli expression system: one from full cDNA sequence and two from fragmentary sequences of Rhodobindin. Hemagglutinating activity and solubility of the recombinant lectins were highest at OD 0.7 cell concentration at 20 °C. The optimized process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.
Assuntos
Assialoglicoproteínas/metabolismo , Sistemas de Liberação de Medicamentos , Fetuínas/metabolismo , Lectinas de Plantas/metabolismo , Rodófitas/química , Alga Marinha/química , Sítios de Ligação , Expressão Gênica , Testes de Hemaglutinação , Fases de Leitura Aberta , Oceano Pacífico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/genética , Lectinas de Plantas/farmacologia , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Controle de Qualidade , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , República da Coreia , Rodófitas/crescimento & desenvolvimento , Alga Marinha/crescimento & desenvolvimento , Solubilidade , Sequências de Repetição em Tandem , TemperaturaRESUMO
BACKGROUNDS: In the present study, we aimed to examine the anti-aging properties of human placental hydrolysate (HPE) and dieckol (DE) from Ecklonia cava against free radical scavenging, muscle hypertrophy-related follistatin mRNA expression, amelioration of cognition-related genes and proteins, inhibition of collagenase-regulating genes, and elastinase activity. METHODS: The anti-aging effects were examined in human fibroblast (CCD986sk), mouse myoblast (C2C12), and neuroblastoma (N2a) cell models, by employing various assays such as 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) scavenging, hydroxyl radical-mediated oxidation, quantitative real-time polymerase chain reaction, enzyme activity, and immunocytochemistry observation. RESULTS: Our results show that HPE combined with DE (HPE:DE) strongly scavenged DPPH radicals and protected proteins against degradation by hydroxyl radical attack. HPE:DE effectively inhibited matrix metalloproteinase-1 expression, protein kinase C alpha expression, and elastinase activity. Furthermore, HPE:DE improved the expression of cognition-related genes (choline acetyltransferase and vesicular acetylcholine transporter). These events may proactively contribute to retard the aging processes and the abrupt physiological changes probably induced by mitochondrial dysfunction with aging. CONCLUSIONS: Based on these findings, we conclude that the combined treatment of HPE:DE may be useful for anti-aging therapy in which the accumulation of oxidative damage is the main driving force.
Assuntos
Envelhecimento/efeitos dos fármacos , Benzofuranos/farmacologia , Phaeophyceae/química , Placenta/química , Hidrolisados de Proteína/farmacologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Linhagem Celular , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99%) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95% of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7%, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and L-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.
Assuntos
Proteínas de Algas/metabolismo , Fertilização , Partenogênese , Phaeophyceae/crescimento & desenvolvimento , Phaeophyceae/metabolismo , Proteínas de Algas/química , Sequência de Aminoácidos , Divisão Celular , Ciclo do Ácido Cítrico , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Dados de Sequência Molecular , Phaeophyceae/citologia , Phaeophyceae/genética , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Succinato-Semialdeído Desidrogenase/química , Succinato-Semialdeído Desidrogenase/metabolismo , Fatores de Tempo , Imagem com Lapso de TempoRESUMO
Cellulose and one dimensional nano-material composite has been investigated for various industrial applications due to their optical, mechanical and electrical properties. In present investigation, cellulose/silica and silica-gold hybrid biomaterials were prepared by sol-gel covalent cross-linking process. The tetraethoxysiliane (TEOS) and gold precursors and γ-aminopropyltriethoxysilane (γ-APTES) as coupling agent were used for sol-gel cross-linking process. The chemical and morphological properties of cellulose/silica and cellulose/silica-gold nano-materials via covalent cross-linking hybrids were confirmed by FTIR, XRD, SEM, and TEM analysis. In the sol-gel process, the inorganic particles were dispersed in the cellulose host matrix at the nanometer scale, bonding to the cellulose through the covalent bonds.
Assuntos
Materiais Biocompatíveis/química , Celulose/química , Eletrônica , Ouro/química , Nanoestruturas/química , Dióxido de Silício/química , Fenômenos Mecânicos , Fenômenos Ópticos , Propilaminas , Silanos/química , TemperaturaRESUMO
Anterior Gradient 2 (AGR2), an ER stress-inducible protein, has been reported to be localized in endoplasmic reticulum (ER) and its level is elevated in numerous metastatic cancers. Recently, it has been demonstrated that AGR2 is involved in the control of ER homeostasis. However, the molecular mechanism how AGR2 regulates ER stress response remains unclear. Herein we show that AGR2 homo-dimerizes through an intermolecular disulfide bond. Moreover, dimerization of AGR2 attenuates ER stress-induced cell death through the association with BiP/GRP78. Thus, these results suggest that dimerization of AGR2 is crucial in mediating the ER stress signaling pathway.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas/metabolismo , Resposta a Proteínas não Dobradas , Apoptose , Linhagem Celular Tumoral , Cisteína/química , Cisteína/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico/química , Humanos , Mucoproteínas , Multimerização Proteica , Proteínas/química , Transdução de SinaisRESUMO
This study was conducted to develop a high-efficiency strain of Spirogyra varians for the production of biomass by radiation breeding. The characteristics of wild-type and mutant S. varians were analyzed through phenomenological and proteomic observations. The results of our phenomenological observations of the S. varians mutant demonstrated increases in growth rate and content of chlorophyll a, b, and a + b; in particular, a significant threefold increase was observed in starch accumulation. Proteomic analysis to investigate the differences in expression between wild-type and mutant proteins identified 18 proteins with significantly different expressions. From the literature review, it was confirmed that the up-regulated proteins were mainly involved in photosynthesis, carbohydrate biosynthesis, and energy metabolism. These results suggest the possibility of algae development by radiation breeding for the production of biofuel.
Assuntos
Raios gama , Mutação , Proteínas de Plantas , Proteoma , Spirogyra , Amido , Clorofila/biossíntese , Clorofila/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteoma/biossíntese , Proteoma/genética , Proteômica/métodos , Spirogyra/genética , Spirogyra/crescimento & desenvolvimento , Spirogyra/metabolismo , Amido/biossíntese , Amido/genéticaRESUMO
The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that the PHOT2 gene is a versatile target gene. We isolated the LsU6-10 promoter from 10 U6 snRNA genes identified from the lettuce genome database for comparison with the AtU6-26 promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the LsU6-10 and AtU6-26 promoters to drive sgRNA361 to target the PHOT2 gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted PHOT2 gene, as in the Arabidopsis phot2 mutant. The PHOT2 gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent phot2 mutants in the R2 generation. Our results suggest that the LsU6-10 promoter is more effective than the AtU6-26 promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that PHOT2 is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes.
RESUMO
In red algae, spermatial binding to female trichogynes is mediated by a lectin-carbohydrate complementary system. Aglaothamnion oosumiense is a microscopic filamentous red alga. The gamete recognition and binding occur at the surface of the hairlike trichogyne on the female carpogonium. Male spermatia are nonmotile. Previous studies suggested the presence of a lectin responsible for gamete recognition on the surface of female trychogynes. A novel N-acetyl-D-galactosamine-specific protein was isolated from female plants of A. oosumiense by affinity chromatography and named AOL1. The lectin was monomeric and did not agglutinate horse blood or human erythrocytes. The N-terminal amino acid sequence of the protein was analyzed, and degenerate primers were designed. A full-length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends-PCR (RACE-PCR). The cDNA was 1,095 bp in length and coded for a protein of 259 amino acids with a deduced molecular mass of 21.4 kDa, which agreed well with the protein data. PCR analysis using genomic DNA showed that both male and female plants have this gene. However, Northern blotting and two-dimensional electrophoresis showed that this protein was expressed 12 to 15 times more in female plants. The lectin inhibited spermatial binding to the trichogynes when preincubated with spermatia, suggesting its involvement in gamete binding.
Assuntos
Lectinas/isolamento & purificação , Rodófitas/química , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cromatografia de Afinidade , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Eletroforese em Gel Bidimensional , Eritrócitos/efeitos dos fármacos , Perfilação da Expressão Gênica , Cavalos , Humanos , Lectinas/química , Lectinas/genética , Dados de Sequência Molecular , Peso Molecular , Rodófitas/genética , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
Background and Objectives: Many preclinical studies have been conducted using animal disease models to determine the effectiveness of human mesenchymal stem cells (hMSCs) for treating immune and inflammatory diseases based on the belief that hMSCs are not immunogenic across species. However, several researchers have suggested xenogeneic immune responses to hMSCs in animals, still without detailed features. This study aimed to investigate a xenogeneic humoral immune response to hMSCs in mice in detail. Methods and Results: Balb/c mice were intraperitoneally injected with adipose tissue-derived or Wharton's jelly-derived hMSCs. Sera from these mice were titrated for each isotype. To confirm specificity of the antibodies, hMSCs were stained with the sera and subjected to a flow cytometic analysis. Spleens were immunostained for proliferating cell nuclear antigen to verify the germinal center formation. Additionally, splenocytes were subjected to a flow cytometric analysis for surface markers including GL-7, B220, CD4, CD8, CD44, and CD62L. Similar experiments were repeated in C57BL/6 mice. The results showed increased IgG1 and IgG2a titers in the sera from Balb/c mice injected with hMSCs, and the titers were much higher in the secondary sera than in the primary sera. These antibodies were specifically stained the hMSCs. Germinal centers were observed in the spleen, and flow cytometric analysis of the splenocytes showed higher frequencies of centroblasts (B220+ GL7+) and memory T cells (CD62L+ CD44+) both in CD4+ and CD8+ subsets. Similar results were obtained for C57BL/6 mice. Conclusions: hMSCs induced a humoral immune response in mice, with characters of T cell-dependent immunity.
RESUMO
In many filamentous red algae, cells that die from physical damage are replaced through somatic fusion of repair cells formed from adjacent cells. We visualized ROS generation in repair cells of Giriffthsia monilis using DCFH-DA staining and examined the expression of the genes involved in wound healing using quantitative PCR. Repair cells elongate along the H2O2 gradient, meet at each other's tips where the H2O2 concentration is highest, and undergo somatic fusion. No wound response occurred with ascorbic acid treatment. Conversely, H2O2 treatment induced many repair cells, leading to multiple somatic cell fusions. Diphenylene iodonium (DPI) or caffeine treatment reversibly inhibited ROS production in repair cells and blocked the progression of the wound response suggesting that ROS and calcium signaling are involved in the process. Four G. monilis homologues of NADPH-oxidase (GmRBOHs) were identified. The expression of GmRBOHs was upregulated upon injury, peaking 1 h post injury, and decreasing to initial levels when repair cells began to elongate. Our results suggest that ROS generated upon cell injury activates Ca2+ channels and upregulates the expression of GmRBOHs, and that H2O2 generated from repair cells mediates induced repair cell elongation leading to somatic cell fusion and filament repair.
Assuntos
Peróxido de Hidrogênio , Rodófitas , Sinalização do Cálcio , Fusão Celular , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Rodófitas/metabolismoRESUMO
Liquid-repellent technology is an efficient means of energy-saving and biofouling avoidance. However, liquid-repellent surfaces suffer from inefficient lubricant retention under shear flow and fouling problem in marine environment. Here, the authors demonstrate a fatty acid amide (FAA)-based oleogel for stable and sustainable lubrication in marine environment. The lubrication management of marine creatures is emulated in synthetic oleogels by incorporating solid (FAA) and liquid lubricants into the molecular meshes of polymeric networks, with the nature-derived solid lubricant providing multifunctional synergistic effects with liquid oil molecules for slippery property and remarkable anti-biofouling. The lubricant-confining gel achieves shear-stable lubricity with efficient oil management. The oleogel provides continued lubrication without biofouling for approximately 4 months in marine field tests. The gel design provides a new paradigm for sustainable and shear-stable lubrication in marine environment.