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1.
Toxins (Basel) ; 16(6)2024 May 21.
Article in English | MEDLINE | ID: mdl-38922129

ABSTRACT

Polyamines (PAs) are polycationic biogenic amines ubiquitously present in all life forms and are involved in molecular signaling and interaction, determining cell fate (e.g., cell proliferation, dif-ferentiation, and apoptosis). The intricate balance in the PAs' levels in the tissues will determine whether beneficial or detrimental effects will affect homeostasis. It's crucial to note that endoge-nous polyamines, like spermine and spermidine, play a pivotal role in our understanding of neu-rological disorders as they interact with membrane receptors and ion channels, modulating neuro-transmission. In spiders and wasps, monoamines (histamine, dopamine, serotonin, tryptamine) and polyamines (spermine, spermidine, acyl polyamines) comprise, with peptides and other sub-stances, the low molecular weight fraction of the venom. Acylpolyamines are venom components exclusively from spiders and a species of solitary wasp, which cause inhibition chiefly of iono-tropic glutamate receptors (AMPA, NMDA, and KA iGluRs) and nicotinic acetylcholine receptors (nAChRs). The first venom acylpolyamines ever discovered (argiopines, Joro and Nephila toxins, and philanthotoxins) have provided templates for the design and synthesis of numerous analogs. Thus far, analogs with high potency exert their effect at nanomolar concentrations, with high se-lectivity toward their ionotropic and ligand receptors. These potent and selective acylpolyamine analogs can serve biomedical purposes and pest control management. The structural modification of acylpolyamine with photolabile and fluorescent groups converted these venom toxins into use-ful molecular probes to discriminate iGluRs and nAchRs in cell populations. In various cases, the linear polyamines, like spermine and spermidine, constituting venom acyl polyamine backbones, have served as cargoes to deliver active molecules via a polyamine uptake system on diseased cells for targeted therapy. In this review, we examined examples of biogenic amines that play an essential role in neural homeostasis and cell signaling, contributing to human health and disease outcomes, which can be present in the venom of arachnids and hymenopterans. With an empha-sis on the spider and wasp venom acylpolyamines, we focused on the origin, structure, derivatiza-tion, and biomedical and biotechnological application of these pharmacologically attractive, chemically modular venom components.


Subject(s)
Insecticides , Polyamines , Spider Venoms , Wasps , Animals , Polyamines/chemistry , Spider Venoms/chemistry , Spider Venoms/toxicity , Insecticides/pharmacology , Insecticides/chemistry , Insecticides/toxicity , Humans , Spiders
2.
Sci Rep ; 14(1): 14945, 2024 Jun 28.
Article in English | MEDLINE | ID: mdl-38942918

ABSTRACT

Particles containing alpha (α) nuclides were identified from sediment in stagnant water in the Unit 3 reactor building of the Fukushima Daiichi Nuclear Power Station (FDiNPS). We analyzed different concentrations of α-nuclide samples collected at two sampling sites, the torus room and the main steam isolation valve (MSIV) room. The solids in the stagnant water samples were classified, and the uranium (U) and total alpha concentrations of each fraction were measured by dissolution followed by inductively coupled plasma mass spectrometry and α-spectrometry. Most of the α-nuclides in the stagnant water samples from the torus and MSIV rooms were in particle fractions larger than 10 µm. We detected uranium-bearing particles ranging from sub-µm to 10 µm in size by scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) observations. The chemical forms of U particles were determined in U-Zr oxides, oxidized UO2, and U3O8 with micro-Raman spectroscopy. Other short-lived α-nuclides (plutonium [Pu], americium [Am], and curium [Cm]) were detected by alpha track detection, and the particles with α-nuclides was characterized by SEM-EDX analysis. α-nuclide-containing particles with several tens to several 100 µm in size mainly comprised iron (Fe) oxyhydroxides. In addition, we detected adsorbed U onto Fe oxyhydroxide particles in the MSIV room sample, which indicated nuclear fuel dissolution and secondary U accumulation. This study clarifies the major characteristics of U and other α-nuclides in sediment in stagnant water in the FDiNPS Unit 3 reactor building, which significantly contribute to the consideration of removal methods for particles containing α-nuclides in the stagnant water.

3.
J Nat Prod ; 87(4): 1159-1170, 2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38552032

ABSTRACT

Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.


Subject(s)
Ascomycota , Ketones , Ascomycota/chemistry , Ascomycota/metabolism , Lactones/chemistry , Lactones/metabolism , Molecular Structure , Ketones/chemistry , Ketones/metabolism
4.
J Nat Prod ; 86(7): 1832-1843, 2023 07 28.
Article in English | MEDLINE | ID: mdl-37385971

ABSTRACT

Paraphaeolactones A1, A2, B1, and B2 (1-4, respectively), known arthropsadiol D (5), massariphenone (6) and its positional isomer 7, and massarilactones E (8) and G (9) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Although the structural resemblance between 1 and 2 implies that these comprised a diastereomeric pair at the C-2 stereogenic center, electronic circular dichroism (ECD) spectral analyses revealed that they were pseudo-enantiomers possessing the common (2R)-configuration. Paraphaeolactones B1 and B2 (3 and 4) were the derivatives of 2, which equipped the 3-(1-hydroxy-2-oxopropyl)-4-methylcatechol moiety via an acetal bond at C-10. The relative configurations of their acetal carbons were elucidated by NOE experiments, and those of C-8' were deduced independently by ECD spectral analysis. The present study disclosed that 1-5, 8, and 9 contain a methylcyclohexene substructure with the same absolute configuration. This prompted us to reinvestigate the absolute configurations of known structurally related fungal metabolites, allowing us to conclude that the methylcyclohexene moieties of these natural products have the same absolute configuration despite the variety of configurations of other stereogenic centers. The plausible biosynthetic routes for 1-9 are discussed on the basis of the above conclusion. We propose a Favorskii rearrangement as the key transformation for biosyntheses of 1-4.


Subject(s)
Acetals , Ascomycota , Lactones , Circular Dichroism , Molecular Conformation , Molecular Structure , Stereoisomerism , Lactones/chemistry
5.
Methods Mol Biol ; 2520: 171-180, 2022.
Article in English | MEDLINE | ID: mdl-35575971

ABSTRACT

The zebrafish is a useful model to identify genes functioning in hematopoiesis, owing to high conservation of hematopoiesis. Flow cytometry is widely used to isolate and quantitatively characterize human and mouse hematopoietic cells, often using fluorescently labeled antibodies. However, such analysis is limited in zebrafish due to lack of antibodies that recognize zebrafish hematopoietic cells. We here describe methods for isolation of hematopoietic cells by antibody-free flow cytometry in the zebrafish embryo. Hematopoietic stem cells (HSCs) are specified from a specific subset of vascular endothelial cells, the hemogenic endothelial cell (HEC), by a high level of Notch signaling. In combination with a Notch reporter line (Tp1:GFP) and a vascular specific line (fli1a:dsRed), HECs can be isolated as Tp1:GFPhigh fli1a:dsRed+ cells at 20-22 hours post-fertilization (hpf). Zebrafish erythrocytes can be purified using 1,5-bis{[2-(dimethylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione (DRAQ5), a DNA-staining fluorescent dye, and gata1:dsRed (erythroid marker line). DRAQ5high dsRed+ cells are morphologically erythrocyte-like, hemoglobin-stained positive, and express erythropoiesis-related genes. Zebrafish neutrophils can be also isolated using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5. PHA-Elow DRAQ5low cells have myeloperoxidase activity, are Sudan Black B-positive, and express neutrophil-related genes. These methods will help to perform genetical and functional assays for various types of hematopoietic cells in zebrafish embryos.


Subject(s)
Flow Cytometry , Hematopoietic Stem Cells , Zebrafish , Animals , Cell Separation/methods , Embryo, Nonmammalian , Endothelial Cells , Erythropoiesis , Flow Cytometry/methods , Hematopoiesis , Zebrafish Proteins
6.
Toxins (Basel) ; 14(4)2022 04 15.
Article in English | MEDLINE | ID: mdl-35448892

ABSTRACT

Arthropods comprise the largest group of living animals, including thousands of species that inhabit marine and terrestrial niches in the biosphere [...].


Subject(s)
Arthropods , Venoms , Animals
7.
Toxins (Basel) ; 13(12)2021 12 10.
Article in English | MEDLINE | ID: mdl-34941722

ABSTRACT

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear α-helical peptides. Among the components of the first class, the two main peptides, α-campsomerin (PRLRRLTGLSPLR) and ß-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only α-campsomerin was able to enhance cell viability, while ß-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of ß-campsomerin, compared to α-campsomerin. Among the linear α-helical peptides, annulatin (ISEALKSIIVG-NH2) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities.


Subject(s)
Insect Proteins/chemistry , Insect Proteins/toxicity , PC12 Cells/drug effects , Toxicological Phenomena/drug effects , Wasp Venoms/chemistry , Wasp Venoms/toxicity , Animals , Japan , Rats
8.
Molecules ; 26(14)2021 Jul 19.
Article in English | MEDLINE | ID: mdl-34299640

ABSTRACT

A series of cyclohumulanoids, i.e., tricocerapicanols A-C (1a-1c), tricoprotoilludenes A (2a) and B (3), tricosterpurol (4), and tricoilludins A-C (5-7) were isolated along with known violascensol (2b) and omphadiol (8) from the culture broth of Daedaleopsis tricolor, an inedible but not toxic mushroom. The structures were fully elucidated on the basis of NMR spectroscopic analysis, and the suggested relative structures were confirmed via density functional theory (DFT)-based chemical shift calculations involving a DP4 probability analysis. In the present study, the 1H chemical shifts were more informative than the 13C chemical shifts to distinguish the diastereomers at C-11. The absolute configurations of 1-5 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. For 6 and 7, the same chirality was assigned according to their biosynthetic similarities with the other compounds. The successful assignment of some Cotton effects was achieved by utilizing DFT calculations using simple model compounds. The plausible biosynthesis of 1-7 was also discussed on the basis of the structural commonality and general cyclohumulanoid biosynthesis. Compounds 2a and 5 were found to simultaneously induce hyphal swelling and branching at 5.0 µg/mL against a test fungus Cochliobolus miyabeanus.


Subject(s)
Culture Media/chemistry , Polyporaceae/growth & development , Sesquiterpenes , Sesquiterpenes/chemistry , Sesquiterpenes/isolation & purification , Sesquiterpenes/pharmacology
9.
J Venom Anim Toxins Incl Trop Dis ; 27: e20200171, 2021 Jun 11.
Article in English | MEDLINE | ID: mdl-34194483

ABSTRACT

BACKGROUND: Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. METHODS: A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. RESULTS: Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and ß-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, ß-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, ß-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. CONCLUSION: Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. ß-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.

10.
Peptides ; 142: 170575, 2021 08.
Article in English | MEDLINE | ID: mdl-34023397

ABSTRACT

Purification of small peptide components in the venoms of the solitary sphecid wasps, Sphex argentatus argentatus and Isodontia harmandi, led to the isolation of several major peptides. Analysis of MS/MS spectra by MALDI-TOF/TOF revealed the sequence of a new peptide Sa112 (EDVDHVFLRF-NH2), which is structurally very similar to leucomyosupressin (pQDVDHVFLRF-NH2) and SchistoFLRFamide (PDVDHVFLRF-NH2), the FMRFamide-like peptides from cockroach and locust, respectively. Indeed, this new peptide, like SchistoFLRFamide, inhibited the frequency and amplitude of spontaneous contractions of the locust oviduct in a dose-dependent manner. A non-amidated peptide Sa12b (EDVDHVFLRF) was also isolated, but this peptide had no effect on spontaneous locust oviduct contraction. This is the first example of a FMRF-like peptide to be found in solitary wasp venom. Additionally, a truncated form of the myosuppressins, which has previously been synthesized and tested for biological activity, DVDHVFLRF-NH2 (Sh5b), was found for the first time as a natural product. Four other novel peptides were isolated and characterized as Sa81 (EDDLEDFNPTVS), Sa10 (EDDLEDFNPTIA), Sh41 (DDLSDFNPKV), and Sh42 (EDDLSDFNPKV). They are structurally related to each other, having a high content of acidic amino acids, but no structural similarity to any known peptides. Ion channel associated activities of Sh41 and Sh42 were tested, but did not show any activity for Na+, K+, Ca2+ channels.


Subject(s)
Locusta migratoria/drug effects , Neuropeptides/isolation & purification , Neuropeptides/pharmacology , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Animals , Female , Oviducts/drug effects , Wasp Venoms/isolation & purification , Wasp Venoms/pharmacology , Wasps
11.
Molecules ; 26(7)2021 Apr 01.
Article in English | MEDLINE | ID: mdl-33915948

ABSTRACT

Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSPCs, the low dose of HSPCs in these preparations limits their use, prompting need for ex vivo HSPC expansion. To establish a more efficient method to expand UCB HSPCs, we developed the bioactive peptide named SL-13R and cultured UCB HSPCs (CD34+ cells) with SL-13R in animal component-free medium containing a cytokine cocktail. Following 9 days of culture with SL-13R, the numbers of total cells, CD34+, CD38- cells, and hematopoietic stem cell (HSC)-enriched cells were significantly increased relative to control. Transplantation of cells cultured with SL-13R into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution and self-renewal ability. AHNAK, ANXA2, and PLEC all interact with SL-13R. Knockdown of these genes in UCB CD34+ cells resulted in reduced numbers of hematopoietic colonies relative to SL-13R-treated and non-knockdown controls. In summary, we have identified a novel bioactive peptide SL-13R promoting expansion of UCB CD34+ cells with long-term reconstitution and self-renewal ability, suggesting its clinical use in the future.


Subject(s)
Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Peptides/pharmacology , Animals , Antigens, CD34/metabolism , Biomarkers , Carrier Proteins , Cell Culture Techniques , Cell Differentiation , Cell Self Renewal , Cells, Cultured , Fluorescent Antibody Technique , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Immunophenotyping , Mice , Protein Binding
12.
J. venom. anim. toxins incl. trop. dis ; 27: e20200171, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1279405

ABSTRACT

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.(AU)


Subject(s)
Animals , Peptides/classification , Wasp Venoms , Wasps/metabolism , Neuroprotection , Oxidative Stress , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
13.
Toxins, v. 13, n. 12, 885, dez. 2021
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-4067

ABSTRACT

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear α-helical peptides. Among the components of the first class, the two main peptides, α-campsomerin (PRLRRLTGLSPLR) and β-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only α-campsomerin was able to enhance cell viability, while β-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of β-campsomerin, compared to α-campsomerin. Among the linear α-helical peptides, annulatin (ISEALKSIIVG-NH2) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities.

14.
J Venom Anim Toxins Incl Trop Dis, v. 27, e20200171, jun. 2021
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3883

ABSTRACT

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.

15.
Genes Cells ; 25(12): 770-781, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33006802

ABSTRACT

Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.


Subject(s)
Flow Cytometry/methods , Hematopoiesis , Neutrophils/cytology , Primary Cell Culture/methods , Animals , Cells, Cultured , Lectins/metabolism , Neutrophils/metabolism , Zebrafish , Zebrafish Proteins/metabolism
16.
Biochem Biophys Res Commun ; 527(3): 730-736, 2020 06 30.
Article in English | MEDLINE | ID: mdl-32439173

ABSTRACT

Fetal liver (FL) is the major embryonic hematopoietic organ and a site where circulating hematopoietic stem/progenitor cells (HSPCs) reside. However, HSPC migration/retention mechanisms in FL remain unclear. A chemokine screen revealed that the CCR4 ligands CCL17 and CCL22 are highly expressed in mouse embryonic day (E) 12.5 FL. Flow cytometric analysis confirmed CCR4 expression in FL HSPCs. To identify sources of CCL17 and CCL22, we fractionated FL into various cell types and found that Ccl17 and Ccl22 were predominantly expressed in HPCs/matured HCs. In vitro cell migration analysis confirmed enhanced HSPC migration in the presence of HPCs/matured HCs. Furthermore, exo-utero injection of anti-CCR4 neutralizing antibody into pregnant mice significantly reduced the number of FL HSPCs in embryos. These data demonstrate a paracrine mechanism by which HSPC migration/retention is regulated by CCL17 and CCL22 secreted from HPCs or matured HCs in FL.


Subject(s)
Chemokine CCL17/metabolism , Chemokine CCL22/metabolism , Hematopoietic Stem Cells/cytology , Liver/embryology , Signal Transduction , Animals , Cell Movement , Cells, Cultured , Hematopoietic Stem Cells/metabolism , Liver/cytology , Mice , Mice, Inbred C57BL , Paracrine Communication
17.
Biol Chem ; 401(8): 945-954, 2020 07 28.
Article in English | MEDLINE | ID: mdl-32229648

ABSTRACT

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that includes one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by online peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g. glutamic acid and proline), free thymidine, and cytosine. To the best of our knowledge, most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptide variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and for defending D. quadriceps against aggressors, predators, and potential microbial infection.


Subject(s)
Ant Venoms/chemistry , Peptides/chemistry , Animals , Ants , Molecular Weight
18.
Toxins (Basel) ; 12(2)2020 01 25.
Article in English | MEDLINE | ID: mdl-31991714

ABSTRACT

Arthropods comprise a predominant and well-succeeded phylum of the animal kingdom that evolved and diversified in millions of species grouped in four subphyla, namely, Chelicerata (arachnids), Crustacea, Myriapoda (centipedes), and Hexapoda (insects) [...].


Subject(s)
Arthropod Venoms , Peptides , Animals , Arthropod Venoms/chemistry , Arthropod Venoms/pharmacology , Arthropod Venoms/therapeutic use , Arthropod Venoms/toxicity , Insecticides/chemistry , Insecticides/pharmacology , Insecticides/therapeutic use , Insecticides/toxicity , Peptides/chemistry , Peptides/pharmacology , Peptides/therapeutic use , Peptides/toxicity
19.
Biol Chem, v. 401, n. 8, p. 945-954, 2020
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-2963

ABSTRACT

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that include one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by on-line peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g., glutamic acid and proline), free thymidine and cytosine. To the best of our knowledge most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptides variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and defending D. quadriceps against aggressors, predators and potential microbial infection.

20.
Toxins (Basel) ; 11(10)2019 10 10.
Article in English | MEDLINE | ID: mdl-31658776

ABSTRACT

In this work, we evaluate the effect of two peptides Sa12b (EDVDHVFLRF) and Sh5b (DVDHVFLRF-NH2) on Acid-Sensing Ion Channels (ASIC). These peptides were purified from the venom of solitary wasps Sphex argentatus argentatus and Isodontia harmandi, respectively. Voltage clamp recordings of ASIC currents were performed in whole cell configuration in primary culture of dorsal root ganglion (DRG) neurons from (P7-P10) CII Long-Evans rats. The peptides were applied by preincubation for 25 s (20 s in pH 7.4 solution and 5 s in pH 6.1 solution) or by co-application (5 s in pH 6.1 solution). Sa12b inhibits ASIC current with an IC50 of 81 nM, in a concentration-dependent manner when preincubation application was used. While Sh5b did not show consistent results having both excitatory and inhibitory effects on the maximum ASIC currents, its complex effect suggests that it presents a selective action on some ASIC subunits. Despite the similarity in their sequences, the action of these peptides differs significantly. Sa12b is the first discovered wasp peptide with a significant ASIC inhibitory effect.


Subject(s)
Acid Sensing Ion Channel Blockers/pharmacology , Acid Sensing Ion Channels/physiology , Ganglia, Spinal/drug effects , Neurons/drug effects , Peptides/pharmacology , Animals , Cells, Cultured , Female , Ganglia, Spinal/physiology , Male , Neurons/physiology , Rats, Long-Evans , Wasps
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