Wild-type AB Research Articles

Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

The emerging contaminant 3,3′-dichlorobiphenyl (PCB-11) impedes Ahr activation and Cyp1a activity to modify embryotoxicity of Ahr ligands in the zebrafish embryo model (Danio rerio)

3,3′-dichlorobiphenyl (PCB-11) is an emerging PCB congener widely detected in environmental samples and human serum, but its toxicity potential is poorly understood. We assessed the effects of three concentrations of PCB-11 on embryotoxicity and Aryl hydrocarbon receptor (Ahr) pathway interactions in zebrafish embryos (Danio rerio). Wildtype AB or transgenic Tg(gut:GFP) strain zebrafish embryos were exposed to static concentrations of PCB-11 (0, 0.2, 2, or 20 μM) from 24 to 96 h post fertilization (hpf), and gross morphology, Cytochrome P4501a (Cyp1a) activity, and liver development were assessed via microscopy. Ahr interactions were probed via co-exposures with PCB-126 or beta-naphthoflavone (BNF). Embryos exposed to 20 μM PCB-11 were also collected for PCB-11 body burden, qRT-PCR, RNAseq, and histology. Zebrafish exposed to 20 μM PCB-11 absorbed 0.18% PCB-11 per embryo at 28 hpf and 0.61% by 96 hpf, and their media retained 1.36% PCB-11 at 28 hpf and 0.84% at 96 hpf. This concentration did not affect gross morphology, but altered the transcription of xenobiotic metabolism and liver development genes, impeded liver development, and increased hepatocyte vacuole formation. In co-exposures, 20 μM PCB-11 prevented deformities caused by PCB-126 but exacerbated deformities in co-exposures with BNF. This study suggests that PCB-11 can affect liver development, act as a partial agonist/antagonist of the Ahr pathway, and act as an antagonist of Cyp1a activity to modify the toxicity of compounds that interact with the Ahr pathway.

Novel diterpenoid-type activators of the Keap1/Nrf2/ARE signaling pathway and their regulation of redox homeostasis

Oxidative stress is involved in the onset and progression of many human diseases. Activators of the Keap1/Nrf2/ARE pathway effectively inhibit the progression of oxidative stress-induced diseases. Herein, a small library of diterpenoids was established by means of phytochemical isolation, and chemical modification on naturally occurring molecules. The diterpenoids were subjected to a NAD(P)H: quinone reductase (QR) assay to evaluate its potential inhibition against oxidative stress. Sixteen diterpenoids were found to be novel potential activators of Nrf2-mediated defensive response. Of which, an isopimarane-type diterpenoid, sphaeropsidin A (SA), was identified as a potent activator of the Keap1/Nrf2/ARE pathway, and displayed approximately 5-folds potency than that of sulforaphane (SF). SA activated Nrf2 and its downstream cytoprotective genes through enhancing the stabilization of Nrf2 in a process involving PI3K, PKC, and PERK, as well as potentially interrupting Nrf2-Keap1 protein-protein interaction. In addition, SA conferred protection against sodium arsenite [As(III)]- and cigarette smoke extract (CSE)-induced redox imbalance and cytotoxicity in human lung epithelial cells, as wells as inhibited metronidazole (MTZ)-induced oxidative insult in Tg (krt4: NTR-hKikGR)cy17 transgenic zebrafish and lipopolysaccharide (LPS)-induced oxidative damage in wild-type AB zebrafish. These results imply that SA is a lead compound for therapeutic agent against oxidative stress-induced diseases, and diterpenoid is a good resource for discovering drug candidates and leads of antioxidant therapy.

Radioprotective effects of mistletoe extract in zebrafish embryos in vivo

Introduction: Radioprotectors can enhance the efficacy of cancer radiotherapy, but their clinical use remains uncommon. The present study aimed to assess the radioprotective potential of mistletoe extract (commercial name: Abnoba Viscum), a well-known complementary cancer medicine, in zebrafish larvae.Materials and methods: Wild-type AB zebrafish embryos at 4 h-post-fertilization were exposed to 5 Gy 9-MeV electron beam irradiation after being treated for 1 h with 4 mMl/L amifostine or 0.2 mg/ml Abnoba Viscum A, F, M, or Q. Primary endpoints were abnormality-free survival and abnormality-free rates among survivors at 5 days-post-fertilization.Results: The crude abnormality-free survival rates were 33.7%, 49.0%, 38.8%, 43.9%, 38.1%, and 52.6%, whereas abnormality-free rates among survivors were 36.4%, 49.6%, 37.8%, 45.6%, 52.0%, and 62.8% for the control (with no pharmacologic treatment), amifostine, Abnoba Viscum A, F, M, and Q groups, respectively. Abnormality-free survival rates in the amifostine and Abnoba Viscum Q groups were significantly different from those in the control (p = .040 and .012, respectively), with an odds ratio (OR) of 1.90 [95% confidence interval (CI): 1.03–3.51] and 2.20 (95% CI: 1.19–4.08), respectively. Abnormality-free rates among survivors in the amifostine and Abnoba Viscum M and Q groups were significantly different from those in the control group (p = .048, .042, and <.001, respectively), with an OR of 1.79 (95% CI: 1.00–3.20), 1.82 (95% CI: 1.02–3.26), and 2.98 (1.67–5.33), respectively.Conclusion: Abnoba Viscum Q has at least a similar radioprotective effect to that of amifostine. Mistletoe extracts have been clinically applied for a long time and their effectiveness and feasibility have been verified. Abnoba Viscum Q might be a new candidate radioprotectant to enhance cancer radiotherapy efficacy.

Transplantation of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors into Early-Stage Zebrafish Embryos.

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.

A Novel Zebrafish (Danio rerio) Assay for Assessing Musk Ambrette-Induced Toxicity.

Musk ambrette (4-tert-butyl-3-methoxy-2,6-dinitrotoluene) is a nitro musk, a cheap substitute for natural musk and a potential environmental pollutant based on its persistence, accumulation in human organisms. We investigated the acute toxicity of musk ambrette using wild-type AB and transgenic Tg(fli1a:EGFP)y1 zebrafish. Different concentrations were delivered to zebrafish by direct soaking from 6 to 72h post-fertilization (hpf). The LC50 of musk ambrette was 76.4µgmL-1. As musk ambrette concentration increased, zebrafish embryos showed developmental delays (50µgmL-1, 22 hpf), pericardial edema (5µgmL-1, 48 hpf), circulatory disturbances, curved body axis (1µgmL-1, 72 hpf) and death (100µgmL-1, 22 hpf). Target organ toxicity was evaluated by a zebrafish angiogenesis model. Musk ambrette induced cardiovascular morphological changes, vessel permeability variation, angiogenic changes and cardiotoxicity (10µgmL-1, 48 hpf). The disappearance of caudal vein plexus confirmed the vascular development toxicity. Musk ambrette negatively affects early life-stage survival and demonstrates various toxicities in zebrafish.

The occhiolino (occ) mutant Zebrafish, a model for development of the optical function in the biological lens.

Zebrafish visual function depends on quality optics. An F3 screen for developmental mutations in the Zebrafish nervous system was conducted in wild-type (wt) AB Zebrafish exposed to 3 mM of N-ethyl-N-nitrosourea (ENU). Mutant offspring, identified in an F3 screen, were characterized by a small pupil, resulting from retinal hypertrophy or hyperplasia and a small lens. Deficits in visual function made feeding difficult after hatching at approximately 5-6 days postfertilization (dpf). Special feeding conditions were necessary for survival of the occhiolino (occ) mutants after 6 dpf. Optokinetic response (OKR) tests measured defects in visual function in the occ mutant, although electroretinograms (ERGs) were normal in the mutant and wt. Consistent with the ERGs, histology found normal retinal structure in the occ mutant and wt Zebrafish. However, lens development was abnormal. Multiphoton imaging of the developmental stages of live embryos confirmed the formation of a secondary mass of lens cells in the developing eye of the mutant Zebrafish at 3-4 dpf, and laminin immunohistochemistry indicated the lens capsule was thin and disorganized in the mutant Zebrafish. The occ Zebrafish is a novel disease model for visual defects associated with abnormal lens development. Developmental Dynamics 246:915-924, 2017. © 2017 Wiley Periodicals, Inc.

Methionine 35 sulphoxide reduces toxicity of Aβ in red blood cell.

The oxidation of methionine residue in position 35 of Ab to sulphoxide (Ab-sulphoxide) has the ability to deeply modify wild-type Ab 1-42 (Ab) neurotoxic action. Our previous studies suggest that in nucleated cells, lower toxicity of Ab-sulphoxide might result not from structural alteration, but from elevation of methionine sulphoxide reductase A (MsrA) activity and mRNA levels. On this basis, we hypothesised that red blood cell (RBC), a cell devoid almost completely of MsrA activity, shares with nucleated cells an antioxidant system induced by methionine 35 sulphoxide, responsible for the lower toxicity of Ab-sulphoxide in RBC. (Results) Supporting this hypothesis, we found that the low toxicity of Ab-sulphoxide in RBC correlated with pentose phosphate pathway (PPP) flux increase, and this event was associated with a low level of methionine oxidation in total proteins. None of these effects were observed when cells were exposed to Ab native. These results outline the importance of the redox state of methionine 35 in the modulation of Ab-mediated events and suggest an important protective role for PPP in RBC of patients affected by Alzheimer's disease.

Polygonum multiflorum root extract as a potential candidate for treatment of early graying hair.

Despite Polygonum multiflorum (PM) has been experiencely used as a drug to treat early graying hair phenomenon in Asian countries for a long time, there is limited study examined the real biological effects of PM on hair graying in vitro and in vivo. In this study, we investigated the effects of PM root extract (PM-RE) on melanin synthesis in human melanoma SKMEL-28 cells and embryos/larvae of wild-type strain AB zebrafish. We also preliminary revealed the molecular mechanism of early hair graying phenomenon in both in vitro and in vivo models. Our results showed that PM-RE significantly induced melanin synthesis in melanin-producing SKMEL-28 melanoma cells and also in zebrafish embryos/larvae at 4-day postfertilization through activation of MC1R/MITF/tyrosinase-signaling pathway. We also investigated the differences in genotype between graying hair follicle and black hair follicle of young peoples and found that early hair graying phenomenon may be related to downregulation of MC1R/MITF/tyrosinase pathway. Taken together, we suggested that PM-RE at safe doses could be used as a potential agent for the treatment of early hair graying and other loss pigmentation-related diseases.

Overexpression of Calr Mutants Perturbs Developmental Hematopoiesis in Zebrafish Embryos

Introduction: Calreticulin (CALR) is a 46-kDa highly conserved, multicompartmental and multifunctional protein. CALR acts as a Ca2+ binding chaperone protein and participates in ensuring proper protein and glycoprotein folding in the endoplasmic reticulum. CALR mutations have been identified in about 30% of JAK2 and MPL unmutated essential thrombocythemia and primary myelofibrosis. Although the expression of CALR mutants resulted in pathogenic thrombocytosis in adult mice, whether CALR mutants may disrupt normal hematopoiesis during early development remains unknown. Here we aimed to evaluate the effects of mutant CALR during embryonic hematopoietic development using the in vivo zebrafish model.Methods: Full-length CALR wild-type,and CALR-del52 and CALR-ins5 mutants cDNAs were subcloned in the pCS2+ vector and a bicistronic pSYC-102 T2A vector, respectively. Capped CALR mRNAs from the above vectors were micro-injected into 1-2 cells stage wild-type AB strain, Tg(cd41:GFP) and Tg(fli1:EGFP) zebrafish embryos, respectively. cd41+ thrombocytes were counted at 3 and 5 days post fertilization (dpf), respectively. Gene expression of hematopoietic lineage-specific and cytokine and cytokine receptor genes were evaluated by quantitative reverse-transcription and real-time polymerase chain reaction (Q-PCR) from 1 to 3 dpf. Morpholino (MO) was used to knock down cytokine receptor genes mpl, epor and csf3r.Results: The expression of CALR proteins from the injection of 100 pg mRNA was confirmed by CALR N-terminal and mutant specific antibodies, respectively. Expression of both CALR-del52 and CALR-ins5 mutant mRNA significantly increased the numbers of hematopoietic stem and progenitor cells in the caudal hematopoietic tissue when compared with CALR-wt mRNA at 3 dpf. No obvious changes in the angiogenesis were visualized in CALR-wt and mutant CALR expressing embryos at 3 dpf in the fli1:EGFP line when compared with uninjected control. Mutant CALR-del52 significantly increased the number of cd41+ thrombocytes at 5 dpf (mean 162.5±4.1 per embryo) when compared to CALR-wt (mean 117.1±3.1 per embryo, P<0.001), mutant CALR-ins5 (mean 128.3±6.1 per embryo, P<0.001) and uninjected control (mean 136.7±3.0 per embryo, P<0.001), respectively.At 5 dpf, the number of cd41+ thrombocytes significantly decreased upon mpl MO knockdown (mean 43.6±4.9 per embryo) when compared to the control MO group (mean 123.5±5.9 per embryo, P<0.001) and the mutant CALR-del52 group (P<0.001). Importantly, co-injection of CALR-del52 mutant mRNA (mean 73.7±5.1 per embryo) can only partially reverse the knockdown effect of mpl MO. In contrast, the numbers of cd41+ thrombocytes did not decrease significantly upon epor MO or csf3r MO knocked-down compared with the control MO group. When CALR-del52 mutant mRNA was co-injected with epor or csf3r MOs, the numbers of cd41+ thrombocytes were comparable to those of CALR-del52-injected embryos.In Q-PCR experiments, the expression of cmyb, runx1 and scl significantly increased in only CALR-ins5 mutant group at 3 dpf when compared to 2 dpf. The expression of αeHb and βeHb significantly decreased in both CALR-del52 and CALR-ins5 mutant groups at 2 dpf but the expression of gata1 remained unchange. The effects of CALR-del52 and CALR-ins5 mutant groups on the expression of cytokine and cytokine receptor genes included the upregulation of mpl at 1 dpf followed by progressive downregulation at 2 dpf and 3 dpf but relatively stable expression of tpo and epo during early development. In the group of genes related to thrombopoiesis, the expression of emilin1a and nbeal2 was significantly downregulated in CALR-del52 group at 3 dpf when compared to 2 dpf.Conclusions: In this study, we showed that the expression of mutant CALR causes thrombocytosis through an mpl-dependent mechanism and perturbs developmental hematopoiesis in zebrafish embryos. DisclosuresNo relevant conflicts of interest to declare.

Cholesteryl hemiesters alter lysosome structure and function and induce proinflammatory cytokine production in macrophages

RationaleCholesteryl hemiesters are oxidation products of polyunsaturated fatty acid esters of cholesterol. Their oxo-ester precursors have been identified as important components of the “core aldehydes” of human atheromata and in oxidized lipoproteins (Ox-LDL). We had previously shown, for the first time, that a single compound of this family, cholesteryl hemisuccinate (ChS), is sufficient to cause irreversible lysosomal lipid accumulation (lipidosis), and is toxic to macrophages. These features, coupled to others such as inflammation, are typically seen in atherosclerosis. ObjectiveTo obtain insights into the mechanism of cholesteryl hemiester-induced pathological changes in lysosome function and induction of inflammation in vitro and assess their impact in vivo. Methods and resultsWe have examined the effects of ChS on macrophages (murine cell lines and primary cultures) in detail. Specifically, lysosomal morphology, pH, and proteolytic capacity were examined. Exposure of macrophages to sub-toxic ChS concentrations caused enlargement of the lysosomes, changes in their luminal pH, and accumulation of cargo in them. In primary mouse bone marrow-derived macrophages (BMDM), ChS-exposure increased the secretion of IL-1β, TNF-α and IL-6. In zebrafish larvae (wild-type AB and PU.1:EGFP), fed with a ChS-enriched diet, we observed lipid accumulation, myeloid cell-infiltration in their vasculature and decrease in larval survival. Under the same conditions the effects of ChS were more profound than the effects of free cholesterol (FC). ConclusionsOur data strongly suggest that cholesteryl hemiesters are pro-atherogenic lipids able to mimic features of Ox-LDL both in vitro and in vivo.

Salvianolic acid B stimulates osteogenesis in dexamethasone-treated zebrafish larvae.

Our previous studies show that salvianolic acid B (Sal B) promotes osteoblast differentiation and matrix mineralization. In this study, we evaluated the protective effects of Sal B on the osteogenesis in dexamethasone (Dex)-treated larval zebrafish, and elucidated the underlying mechanisms. At 3 d post fertilization, wild-type AB zebrafish larvae or bone transgenic tg (sp7:egfp) zebrafish larvae were exposed to Sal B, Dex, or a mixture of Dex+Sal B for 6 d. Bone mineralization in AB strain larval zebrafish was assessed with alizarin red staining, and osteoblast differentiation in tg (sp7:egfp) larval zebrafish was examined with fluorescence scanning. The expression of osteoblast-specific genes in the larvae was detected using qRT-PCR assay. The levels of oxidative stress markers (ROS and MDA) in the larvae were also measured. Exposure to Dex (5-20 μmol/L) dose-dependently decreased the bone mineralization area and integral optical density (IOD) in wild-type AB zebrafish larvae and the osteoblast fluorescence area and IOD in tg (sp7:egfp) zebrafish larvae. Exposure to Dex (10 μmol/L) significantly reduced the expression of osteoblast-specific genes, including runx2a, osteocalcin (OC), alkaline phosphatase (ALP) and osterix (sp7), and increased the accumulation of ROS and MDA in the larvae. Co-exposure to Sal B (0.2-2 μmol/L) dose-dependently increased the bone mineralization area and IOD in AB zebafish larvae and osteoblast fluorescence in tg (sp7:egfp) zebrafish larvae. Co-exposure to Sal B (2 μmol/L) significantly attenuated deleterious alterations in bony tissue and oxidative stress in both Dex-treated AB zebafish larvae and tg (sp7:egfp) zebrafish larvae. Sal B stimulates bone formation and rescues GC-caused inhibition on osteogenesis in larval zebrafish by counteracting oxidative stress and increasing the expression of osteoblast-specific genes. Thus, Sal B may have protective effects on bone loss trigged by GC.

Phytoestrogen Bakuchiol Exhibits In Vitro and In Vivo Anti-breast Cancer Effects by Inducing S Phase Arrest and Apoptosis.

Phytoestrogen has been proposed as an alternative to hormone replacement therapy, which has been demonstrated to promote a high risk of breast cancer. However, the effect of phytoestrogen on breast cancer development has not been fully understood. Bakuchiol is an active ingredient of a traditional Chinese herbal medicine Fructus Psoraleae, the dried ripe fruit of Psoralea corylifolia L. (Fabaceae). The in vitro and in vivo estrogenic activities and anti-breast cancer effects of bakuchiol have not been well-studied. We found that bakuchiol induced the GFP expression in transgenic medaka (Oryzias melastigma, Tg, Chg:GFP) dose-dependently (0–1 μg/ml), demonstrating its in vivo estrogenic activity. Low dose of bakuchiol (1 μg/ml) induced the cell proliferation and ERα expression in MCF-7 cells, which could be blocked by the anti-estrogen ICI 182780, suggesting the in vitro estrogenic activity of bakuchiol. Our data indicated that high doses of bakuchiol (>2 μg/ml) inhibited breast cancer cell growth, with a stronger anti-proliferative effect than resveratrol, a widely studied analog of bakuchiol. High doses of bakuchiol (4, 7, and 10 μg/ml) were used for the further in vitro anti-breast cancer studies. Bakuchiol induced ERβ expression and suppressed ERα expression in MCF-7 cells. It also induced S phase arrest in both MCF-7 and MDA-MB-231 cells, which could be rescued by caffeine. Knock-down of p21 also marginally rescued S phase arrest in MCF-7 cells. The S phase arrest was accompanied by the upregulation of ATM, P-Cdc2 (Tyr15), Myt1, P-Wee1 (Ser642), p21 and Cyclin B1, suggesting that blocking of Cdc2 activation may play an important role in bakuchiol-induced S phase arrest. Furthermore, bakuchiol induced cell apoptosis and disturbed mitochondrial membrane potential in MCF-7 cells. The bakuchiol-induced apoptosis was associated with increased expression of Caspase family and Bcl-2 family proteins, suggesting that bakuchiol may induce apoptosis via intrinsic apoptotic pathway. The in vivo anti-breast cancer effect of bakuchiol was further proved in zebrafish (Danio rerio, wild-type AB) xenografts. 0.5 μg/ml of bakuchiol significantly reduced the MCF-7 cell mass in zebrafish xenografts. Overall, these results suggested the potential of using bakuchiol in HRT and breast cancer treatment.

Hearing Assessment in Zebrafish During the First Week Postfertilization.

The zebrafish (Danio rerio) is a valuable vertebrate model for human hearing disorders because of many advantages in genetics, embryology, and in vivo visualization. In this study, we investigated auditory function of zebrafish during the first week postfertilization using microphonic potential recording. Extracellular microphonic potentials were recorded from hair cells in the inner ear of wild-type AB and transgenic Et(krt4:GFP)(sqet4) zebrafish at 3, 5, and 7 days postfertilization in response to 20, 50, 100, 200, 300, and 400-Hz acoustic stimulation. We found that microphonic threshold significantly decreased with age in zebrafish. However, there was no significant difference of microphonic responses between wild-type and transgenic zebrafish, indicating that the transgenic zebrafish have normal hearing like wild-type zebrafish. In addition, we observed that microphonic threshold did not change with the recording electrode location. Furthermore, microphonic threshold increased significantly at all tested stimulus frequencies after displacement of the saccular otolith but only increased at low frequencies after displacement of the utricular otolith, showing that the saccule rather than the utricle plays the major role in larval zebrafish hearing. These results enhance our knowledge of early development of auditory function in zebrafish and the factors affecting hearing assessment with microphonic potential recording.

Changes in zebrafish phagocytic activity induced by acute ethanol exposure vary by time of day and leukocyte type (INC5P.327)

Abstract Chronic alcohol abuse and acute alcohol consumption (binge drinking) have been associated with suppression of immune function. Reduced phagocytic activity in macrophages and altered levels of inflammatory mediators have been reported in mammalian studies of ethanol exposure. To explore whether the zebrafish model could be valuable in understanding the effects of ethanol on innate immune activity, we assessed the activity of zebrafish phagocytes after acute exposure to ethanol in tank water. Experiments were conducted at different times of day to also determine whether effects of ethanol vary in a diurnal pattern. Treating wild-type AB strain fish with ethanol in the morning reduced the percentage of total phagocytic cells that ingested bacteria. The opposite results were observed in the evening, when a higher proportion of cells participated in phagocytosis compared to controls. To distinguish between the activity of macrophages and neutrophils, mpx:GFP zebrafish were also used. Exposure to ethanol in tank water resulted in a reduction in the amount of bacteria engulfed by neutrophils, whereas changes in macrophage phagocytic activity were not as evident. These initial results indicate that ethanol affects subsets of phagocytic cells differently, and that the effects may be time-of-day dependent.

A Convenient Dry Feed for Raising Zebrafish Larvae

Five protocols for raising zebrafish larvae were tested and compared in this study. These include four variations of feeding Fry Feed Kyowa N-250 (Kyowa) obtained from Miyako Kagaku, as well as an established protocol using Paramecia and supplemented with Zeigler dry feeds. The performance of these feeding protocols was evaluated using larvae obtained from two wild-type strains AB and AB/TL and two mutant strains gassi and smarca4(a50). Several quality parameters including survival rate, rearing density, growth rate, sex ratio, fecundity, eggs spawned by the resulting fish, and the corresponding survival rate were compared by logistic regression analyses. The results showed that the quality of AB and AB/TL larvae raised using one of the Kyowa feeding protocols was equally good when compared with the standard Paramecium protocol for up to 20 larvae/L. The protocol also provided a comparable but slightly lower survival rate for smarca4(a50) and inbred gassi lines, respectively. Nonetheless, all other quality parameters in the case of gassi were comparable to the established Paramecium protocol, even though the line carried multiple pigmentation mutations. Therefore, the optimized Kyowa feeding protocol can substantially reduce routine workload in maintaining a zebrafish research facility by virtually eliminating the need to culture live Paramecia feed.

Directing the natural catalytic activity of antibodies to hepatitis C virus E2 protein (47.36)

Abstract The envelope protein E2 is involved in hepatitis C virus (HCV) entry into host cells. The proteolytic activity of antibodies (Abs) offers a novel neutralizing mechanism by permanent inactivation of multiple epitopes by a single Ab molecule. We studied the proteolytic cleavage of E2 linked to a glutathione S-transferase tag (E2-GST) by Abs purified from human sera. IgM but not IgG class Abs hydrolyzed recombinant E2 determined by an electrophoresis assay, consistent with previous observations that Ab catalysis is an innate immune function subject to deterioration over the course of adaptive B lymphocyte differentiation. To determine the feasibility of engineering HCV E2-specific proteolytic Abs, we cloned hybrid human IgGs composed of light chains with promiscuous proteolytic activity (clones GG63, HK13 and HK14; isolated from a lupus phage library) and the heavy chain of an HCV E2-specific Ab (clone CBH-7; isolated from an HCV infected subject). The hybrid Abs hydrolyzed E2-GST with little or no activity in the wildtype control Ab. The hybrid Abs displayed the ability to bind 125I-E2, confirming that the heavy chain subunit can imparts specificity for E2. These observations suggest the feasibility of harnessing the natural proteolytic activity of Abs to developing catalytic reagents with potential immunotherapeutic utility.

An Engineered Human IgG1 Antibody with Longer Serum Half-Life

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was approximately 2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.

Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect.

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.

Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft

Penicillin G acylase is a periplasmic protein, cytoplasmically expressed as a precursor polypeptide comprising a signal sequence, the A and B chains of the mature enzyme (209 and 557 residues respectively) joined by a spacer peptide of 54 amino acid residues. The wild-type AB heterodimer is produced by proteolytic removal of this spacer in the periplasm. The first step in processing is believed to be autocatalytic hydrolysis of the peptide bond between the C-terminal residue of the spacer and the active-site serine residue at the N terminus of the B chain. We have determined the crystal structure of a slowly processing precursor mutant (Thr263Gly) of penicillin G acylase from Escherichia coli, which reveals that the spacer peptide blocks the entrance to the active-site cleft consistent with an autocatalytic mechanism of maturation. In this mutant precursor there is, however, an unexpected cleavage at a site four residues from the active-site serine residue. Analyses of the stereochemistry of the 260–261 bond seen to be cleaved in this precursor structure and of the 263–264 peptide bond have suggested factors that may govern the autocatalytic mechanism.