scholarly journals Molecular Identification and Dual Functions of Two Different CXC Chemokines in Nile Tilapia (Oreochromis niloticus) against Streptococcus agalactiae and Flavobacterium columnare

2020 ◽  
Vol 8 (7) ◽  
pp. 1058
Author(s):  
Chatsirin Nakharuthai ◽  
Prapansak Srisapoome
Keyword(s):  
Streptococcus Agalactiae ◽  
Nile Tilapia ◽  
Time Course ◽  
Head Kidney ◽  
Flavobacterium Columnare ◽  
Typical Structure ◽  
Cxc Chemokine ◽  
Time Course Experiment ◽  
Functional Analyses

Two CXC chemokines in Nile tilapia (On-CXC1 and On-CXC2) were identified at both the genomic and proteomic levels. A southern blot analysis and comparison searching in Ensembl confirmed the typical structure of the CXC chemokine genes and provided evidence for unusual mechanisms used to generate the two different CXC chemokine transcripts that have not been reported in other vertebrate species so far. The expression levels of On-CXC1 and On-CXC2 were analyzed by quantitative real-time PCR. These two mRNAs were detected in various tissues of normal Nile tilapia, especially in the spleen, heart, and head kidney, indicating a homeostatic function in immunosurveillance. A time-course experiment clearly demonstrated that these two transcripts were effectively enhanced in the head kidney, spleen and trunk kidney of Nile tilapia 6, 12 and 24 h after injection with Streptococcus agalactiae but were down-regulated in all tested tissues at 48 h, reflecting the fact that they have short half-lives during the crucial response to pathogens that is characteristic of CXC chemokine genes in other vertebrates. Functional analyses obviously exhibited that these two CXC chemokines at concentrations of 1–10 μg strongly inactivated S. agalactiae and Flavobacterium columnare and effectively induced phagocytosis of leukocytes in vitro.

scholarly journals Immune Regulation, but Not Antibacterial Activity, Is a Crucial Function of Hepcidins in Resistance against Pathogenic Bacteria in Nile Tilapia (Oreochromis niloticus Linn.)

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1132
Author(s):  
Pagaporn Phan-Aram ◽  
Gunanti Mahasri ◽  
Pattanapon Kayansamruaj ◽  
Piti Amparyup ◽  
Prapansak Srisapoome
Keyword(s):  
Immune Regulation ◽  
Nile Tilapia ◽  
Pathogenic Bacteria ◽  
Mononuclear Cells ◽  
Head Kidney ◽  
Antibacterial Activities ◽  
Phagocytic Index ◽  
Peripheral Blood Mononuclear ◽  
Nile Tilapia Oreochromis Niloticus

In this study, the functions of a recombinant propeptide (rProOn-Hep1) and the synthetic FITC-labelled mature peptides sMatOn-Hep1 and sMatOn-Hep2 were analyzed. Moreover, sMatOn-Hep1 and sMatOn-Hep2 were mildly detected in the lymphocytes of peripheral blood mononuclear cells (PBMCs) and strongly detected in head kidney macrophages. The in vitro binding and antibacterial activities of these peptides were slightly effective against several pathogenic bacteria. Immune regulation by sMatOn-Hep1 was also analyzed, and only sMatOn-Hep1 significantly enhanced the phagocytic index in vitro (p < 0.05). Interestingly, intraperitoneal injection of sMatOn-Hep1 (10 or 100 µg) significantly elevated the phagocytic activity, phagocytic index, and lysozyme activity and clearly decreased the iron ion levels in the livers of the treated fish (p < 0.05). Additionally, sMatOn-Hep1 enhanced the expression levels of CC and CXC chemokines, transferrin and both On-Hep genes in the liver, spleen and head kidney, for 1–96 h after injection, but did not properly protect the experimental fish from S. agalactiae infection after 7 days of treatment. However, the injection of S. agalactiae and On-Heps indicated that 100 μg of sMatOn-Hep1 was very effective, while 100 μg of rProOn-Hep1 and sMatOn-Hep2 demonstrated moderate protection. Therefore, On-Hep is a crucial iron-regulating molecule and a key immune regulator of disease resistance in Nile tilapia.

scholarly journals Ubc4/5 and c-Cbl Continue to Ubiquitinate EGF Receptor after Internalization to Facilitate Polyubiquitination and Degradation

2008 ◽  
Vol 19 (8) ◽  
pp. 3454-3462 ◽  
Author(s):  
Kyohei Umebayashi ◽  
Harald Stenmark ◽  
Tamotsu Yoshimori
Keyword(s):  
Ubiquitin Ligase ◽  
Time Course ◽  
Kinase Activity ◽  
Egf Receptor ◽  
In Vitro Activity ◽  
Lysosomal Sorting ◽  
Time Course Experiment ◽  
Ubiquitin Conjugating Enzyme ◽  
Epidermal Growth

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.

Quantitative analysis in LC3-II protein in vitro maturation of porcine oocyte

Zygote ◽  
2013 ◽  
Vol 22 (3) ◽  
pp. 404-410 ◽  
Author(s):  
SeungHoon Lee ◽  
Yuuki Hiradate ◽  
Yumi Hoshino ◽  
Kentaro Tanemura ◽  
Eimei Sato
Keyword(s):  
Western Blotting ◽  
Time Course ◽  
Cumulus Cells ◽  
Lysosomal Protease ◽  
Time Course Experiment ◽  
Porcine Oocyte ◽  
Autophagic Degradation ◽  
Lysosomal Proteases ◽  
Pepstatin A

SummaryMicrotubule-associated protein light chain 3 (LC3)-II is a marker of autophagosome. In this study, LC3-II expression was used to identify autophagy, during the in vitro maturation of porcine oocytes. In a time-course experiment, cumulus–oocyte complexes (COCs) were cultured in NCSU23 medium for 0 h, 14 h, 28 h or 42 h. The cumulus cells were removed and denuded oocytes were processed for western blotting or immunostaining. Western blotting showed that the LC3-II levels changed over time, with maximum levels observed at 14 h and minimum levels at 42 h. Immunostaining of LC3 showed the signals with dot shapes and ring shapes in oocytes at every group that probably represent autophagosomes. To ascertain whether autophagic induction and degradation were occurring, we treated the cultures with autophagic inhibitors. Lysosomal protease inhibitor E64d and pepstatin A increased the LC3-II levels and wortmannin, inhibitor of autophagic induction, decreased the LC3-II levels. Western blotting and immunostaining demonstrated that LC3-II is present in porcine oocytes cultured in vitro. The decreased LC3-II levels after wortmannin treatment suggest that it is newly generated in porcine oocytes, a phenomenon that represents autophagic induction. Furthermore, increased LC3-II levels after E64d and pepstatin A addition imply that LC3-II is degraded by lysosomal proteases, an indication of autophagic degradation. Our results suggest that autophagy, which is a dynamic process whereby autophagosomes are newly generated and subsequently degraded, is probably occurring in porcine oocytes during in vitro maturation.

scholarly journals Capitalizing on transcriptome profiling to optimize and identify targets for promoting early murine folliculogenesis in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrea Jones ◽  
Beatriz Peñalver Bernabé ◽  
Vasantha Padmanabhan ◽  
Jun Li ◽  
Ariella Shikanov
Keyword(s):  
Childhood Cancer ◽  
Time Course ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Transcriptome Profiling ◽  
Culture Methods ◽  
Time Course Experiment ◽  
Underlying Mechanisms ◽  
Survivors Of Childhood Cancer

AbstractIn vitro ovarian follicle culture is an active area of research towards providing fertility options for survivors of childhood cancer. Late-stage murine follicles (multilayer secondary and onwards) can be cultured successfully to maturity to obtain a meiotically competent oocyte for fertilization, but primordial and primary follicles usually die in culture because many key components of early follicle development are still unknown and difficult to mimic in vitro. To engineer a biomimetic three-dimensional culture system with high efficacy and reproducibility for the clinic, detailed mechanisms of early folliculogenesis must be uncovered. Previous studies have shown that primary murine follicles co-cultured in groups, in contrast to single follicles cultured in isolation, can reach preovulatory size and produce competent oocytes, but the factors accounting for the synergy of follicle co-culture are still unknown. To probe the underlying mechanisms of successful follicle co-culture, we conducted a time-course experiment for murine follicles encapsulated in 0.3% alginate hydrogels and compared between two conditions: groups of 5 (5X) versus groups of 10 (10X). For every 2 days during the course of 12 days, follicles were dissociated and somatic cells were isolated for microarray-based gene expression analysis (n = 380 follicles for 5X and n = 430 follicles for 10X). Gene activities in follicles co-cultured in larger groups (10X) had a distinct transcriptomic profile of key genes and pathways such as prolactin signaling and angiogenesis-related genes when compared to cells from follicles co-cultured in the smaller cohort (5X). To benchmark the results for follicles grown in culture, we compared our microarray data to data from murine follicles freshly isolated from the ovary at comparable stages of development previously published by Bernabé et al. Comparison of these datasets identified similarities and differences between folliculogenesis in the native microenvironment and the engineered in vitro system. A more detailed understanding of follicle growth in vitro will not only allow for better culture methods but also advance the field towards providing improved fertility options for survivors of childhood cancer.

scholarly journals LECT2 Protects Nile Tilapia (Oreochromis niloticus) Against Streptococcus agalatiae Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Qi Li ◽  
Zhiqiang Zhang ◽  
Weiqi Fan ◽  
Yongxiong Huang ◽  
Jinzhong Niu ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacterial Infection ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Head Kidney ◽  
Transcriptional Level ◽  
Reading Frame ◽  
Tissue Damages

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that especially plays an important role in innate immune. However, the roles of LECT2 in the immune response of the economically important fish Nile tilapia (Oreochromis niloticus) against bacterial infection remains unclear. In this study, a lect2 gene from Nile tilapia (On-lect2) was identified, and its roles in the fish’s immune response against bacterial infection were determined and characterised. On-lect2 contains an open reading frame of 456 bp that encodes a peptide of 151 amino acids, as well as the conservative peptidase M23 domain. On-LECT2 is 62%–84% identical to other fish species and about 50% identical to mammals. The highest transcriptional level of On-lect2 was detected in the liver, whereas the lowest levels were detected in the other tissues. Moreover, the On-LECT2 protein is located mainly in the brain and head kidney. The transcriptional levels of On-lect2 substantially increased in the head kidney, brain, liver and spleen after Streptococcus agalactiae infection. Knockdown On-lect2 led to higher mortality due to liver necrosis or haemorrhage and splenomegaly. In vitro analysis indicated that the recombinant protein of On-LECT2 improved phagocytic activity of head kidney-derived macrophages. In vivo challenge experiments revealed several functions of On-LECT2 in the immune response of Nile tilapia against bacterial infection, including promotion of inflammation, reduction of tissue damages and improvement of survival rate.

scholarly journals Differences in Virulence of Marine and Freshwater Isolates of Viral Hemorrhagic Septicemia Virus In Vivo Correlate with In Vitro Ability To Infect Gill Epithelial Cells and Macrophages of Rainbow Trout (Oncorhynchus mykiss)

2008 ◽  
Vol 82 (21) ◽  
pp. 10359-10365 ◽  
Author(s):  
Bjørn E. Brudeseth ◽  
Helle F. Skall ◽  
Øystein Evensen
Keyword(s):  
Rainbow Trout ◽  
Epithelial Cells ◽  
Oncorhynchus Mykiss ◽  
Time Course ◽  
Primary Cultures ◽  
Head Kidney ◽  
Viral Hemorrhagic Septicemia Virus ◽  
Viral Hemorrhagic Septicemia

ABSTRACT Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species.

scholarly journals Opportunities to improve nitrogen utilisation in the rumen by reduction in plant-mediated proteolysis

2007 ◽  
pp. 187-192
Author(s):  
B.A. Barrett ◽  
D. Pacheco ◽  
W.C. Mcnabb ◽  
H.S. Easton
Keyword(s):  
Perennial Ryegrass ◽  
Free Amino ◽  
Time Course ◽  
N Loss ◽  
Total N ◽  
Rumen Microbes ◽  
Time Course Experiment ◽  
Series Of Experiments ◽  
In Sacco

The reaction of ingested forage plant fragments to the rumen environment may contribute to the rapid degradation of plant cell contents in the rumen. A series of experiments were used to explore the hypothesis that plant-mediated proteolysis (PMP) contributes to N loss from fresh forage entering the rumen, and to investigate the potential for identifying cultivars with reduced PMP. During an in sacco time-course experiment, chopped fresh field grown perennial ryegrass (Lolium perenne L.) leaf samples lost 15% of their total N within the first 8 hours in the absence of rumen microbes; whereas 40% was lost from samples incubated with exposure to rumen microbes. Accumulation of free amino acids in the buffer accounted for 75% of the N loss after 24 hours. In a subsequent experiment, in vitro incubation of samples from five perennial ryegrass cultivars detected significant (P

scholarly journals Sarcodia suieae Acetyl-Xylogalactan Regulates Nile Tilapia (Oreochromis niloticus) Tissue Phagocytotic Activity and Serum Indices

2021 ◽  
Vol 10 (1) ◽  
pp. 18
Author(s):  
Po-Kai Pan ◽  
Tsung-Meng Wu ◽  
Chiu-Ming Wen ◽  
Yin-Yu Chen ◽  
Yu-Sheng Wu
Keyword(s):  
Glucose Uptake ◽  
Phagocytic Activity ◽  
Nile Tilapia ◽  
Macrophage Activation ◽  
Head Kidney ◽  
Macrophage Polarisation ◽  
In Vivo Experiments ◽  
Blood Biochemical

Sarcodia suieae acetyl-xylogalactan was reported to induce macrophage polarisation, and could positively regulate macrophage activation. In this study, we evaluated the effect of Sarcodia suieae acetyl-xylogalactan on the Nile tilapia. First, we assessed the influence of acetyl-xylogalactan on the survival, glucose uptake, and phagocytic activity of tilapia head kidney (THK) melanomacrophage, and observed increased proliferation of these cells in the MTT assay after 12 and 24 h of treatment. Glucose uptake increased in THK melanomacrophage treated with 20 and 30 μg acetyl-xylogalactan for 24 h. Their phagocytic activity was positively enhanced following exposure to acetyl-xylogalactan. Nile tilapia were fed with acetyl-xylogalactan for 4 weeks. At the end of the experiment, Nile tilapia were sacrificed, and the lipopolysaccharide-induced liver and head-kidney apoptosis was examined under reducing conditions in comparison with controls. The phagocytic activities of liver and head-kidney cells were enhanced after 4 weeks of feeding. Blood biochemical analysis revealed a reduction in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels after 4 weeks of feeding. Combined with in vitro and in vivo experiments results, the extracted S. suieae acetyl-xylogalactan could directly induce THK melanomacrophage proliferation, glucose uptake, and phagocytic activity. Acetyl-xylogalactan was able to induce Nile tilapia liver and head-kidney resident macrophage activity, and reduced LPS-induced liver and head-kidney cell apoptosis. S. suieae acetyl-xylogalactan may modulate Nile tilapia macrophage activation by polarising them into M1 macrophages to improve the Nile tilapia nonspecific immune response.

Antimicrobial Properties of the Chelating Agent EDTA on Streptococcal Bovine Mastitis Isolates

2006 ◽  
Vol 69 (6) ◽  
pp. 1460-1462 ◽  
Author(s):  
JEFFREY S. REIDMILLER ◽  
WAYNE L. SMITH ◽  
MARY M. SAWYER ◽  
BENNIE I. OSBURN ◽  
JEFFERY L. STOTT ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Time Course ◽  
Microbial Growth ◽  
Bovine Mastitis ◽  
Antimicrobial Properties ◽  
Chelating Agent ◽  
Experimental Conditions ◽  
Bacterial Cultures ◽  
Streptococcus Uberis

To determine the efficacy of the chelating agent EDTA on microbial growth, separate cultures of two streptococcal bovine mastitis isolates, Streptococcus agalactiae and Streptococcus uberis, were exposed to known concentrations of EDTA. Bacterial cultures of 108 CFU/ml were exposed to concentrations of EDTA ranging from 30 to 100 mM in an in-vitro-milk environment. Multiple replications of cultures exposed to EDTA were plated during a two-hour time course. A concentration of 100 mM EDTA resulted in a 90% reduction of S. agalactiae and a 99% reduction of S. uberis. Under these experimental conditions, EDTA treatments in cultures of both isolates exhibited from 1 to 2 log reductions suggesting that EDTA is a potentially effective antimicrobial against streptococcal isolates implicated in causing bovine mastitis.

In vitro and in vivo screening of herbal extracts against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Aquaculture ◽  
2019 ◽  
Vol 503 ◽  
pp. 412-421 ◽  
Author(s):  
Wei-Liang Guo ◽  
Heng-Wei Deng ◽  
Fei Wang ◽  
Shi-Feng Wang ◽  
Zhi-Hong Zhong ◽  
...  
Keyword(s):  
Streptococcus Agalactiae ◽  
Oreochromis Niloticus ◽  
Nile Tilapia ◽  
Herbal Extracts ◽  
In Vivo Screening ◽  
Nile Tilapia Oreochromis Niloticus
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