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1.
Int J Mol Sci ; 24(16)2023 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-37629088

RESUMO

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.


Assuntos
Exossomos , Vesículas Extracelulares , Holothuria , Pepinos-do-Mar , Animais , Evolução Biológica , Vesícula , Mamíferos
2.
Parasitol Int ; 91: 102647, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35985636

RESUMO

A series of 1-aryl-4-(phthalimidoalkyl) piperazines and 1-aryl-4-(naphthalimidoalkyl) piperazines were retrieved from a proprietary library based on their high structural similarity to haloperidol, an antipsychotic with antiparasitic activity, and assessed as potential antileishmanial scaffolds. Selected compounds were tested for antileishmanial activity against promastigotes of Leishmania major and Leishmania mexicana in dose-response assays. Two of the 1-aryl-4-(naphthalimidoalkyl) piperazines (compounds 10 and 11) were active against promastigotes of both Leishmania species without being toxic to human fibroblasts. Their activity was found to correlate with the length of their alkyl chains. Further analyses showed that compound 11 was also active against intracellular amastigotes of both Leishmania species. In promastigotes of both Leishmania species, compound 11 induced collapse of the mitochondrial electrochemical potential and increased the intracellular Ca2+ concentration. Therefore, it may serve as a promising lead compound for the development of novel antiparasitic drugs.


Assuntos
Antiprotozoários , Leishmania major , Leishmania mexicana , Antiparasitários , Antiprotozoários/química , Antiprotozoários/farmacologia , Humanos , Piperazinas/farmacologia
3.
Int J Mol Sci ; 23(12)2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35743119

RESUMO

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.


Assuntos
Pepinos-do-Mar , Animais , Desoxirribonucleases/metabolismo , Endopeptidases/metabolismo , Humanos , Metaloproteases/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Pepinos-do-Mar/metabolismo
4.
Int J Hematol ; 116(2): 192-198, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35610497

RESUMO

Enucleation is a crucial event during the erythropoiesis, implicating drastic morphologic and transcriptomic/proteomic changes. While many genes deletion lead to failed or impaired enucleation have been identified, directly triggering the erythroid maturation, particularly enucleation, is still challenging. Inducing enucleation at the desired timing is necessary to develop efficient methods to generate mature, fully functional red blood cells in vitro for future transfusion therapies. However, there are considerable differences between primary erythroid cells and cultured cell sources, particularly pluripotent stem cell-derived erythroid cells and immortalized erythroid cell lines. For instance, the difference in the proliferative status between those cell types could be a critical factor, as cell cycle exit is closely connected to the terminal maturation of primary. In this review, we will discuss previous findings on the enucleation machinery and current challengings to trigger the enucleation of infinite erythroid cell sources.


Assuntos
Células-Tronco Pluripotentes , Proteômica , Diferenciação Celular/genética , Eritrócitos , Células Eritroides , Eritropoese/genética , Humanos
5.
Molecules ; 27(6)2022 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-35335321

RESUMO

Metal ions and other elements play many different critical roles in all biological processes. They can be especially important in high concentrations for the functioning of organisms living in seawater. It is important to understand how much the concentrations of different trace elements in such organisms can be higher than in seawater. Some marine organisms capable of rapid recovery after different injuries are fascinating in this regard. Sea cucumbers Eupentacta fraudatrix can completely restore all organs and the whole body within several weeks after their division into two parts. Here, for the first time, a comparison of the content of different elements in seawater, sea cucumber, and its very stable multiprotein complex (2000 kDa) was performed using two-jet plasma atomic emission spectrometry. Among the 18 elements we found in sea cucumbers, seawater contained only six elements in detectable amounts, and their content decreased in the following order: Mg > Ca > B > Sr ≈ Si > Cr (0.13−930 µg/g of seawater). The content of these elements in sea cucumbers was higher compared with seawater (-fold): Ca (714) > Sr (459) > Cr (75) > Si (42)> B (12) > Mg (6.9). Only four of them had a higher concentration in the protein complex than in seawater (-fold): Si (120.0) > Cr (31.5) > Ca (9.1) > Sr (8.8). The contents of Mg and B were lower in the protein complex than in seawater. The content of elements additionally found in sea cucumbers decreased in the order (µg/g of powder) of P (1100) > Fe (47) > Mn (26) > Ba (15) > Zn (13) > Al (9.3) > Mo (2.8) > Cu (1.4) > Cd (0.3), and in the protein complex, in the order of P (290) > Zn (51) > Fe (23) > Al (14) ≈ Ni (13) > Cu (7.5) > Ba (2.5) ≈ Co (2.0) ≈ Mn (1.6) > Cd (0.7) >Ag (0.2). Thus, sea cucumbers accumulate various elements, including those contained in very low concentrations in seawater. The possible biological roles of these elements are discussed here.


Assuntos
Pepinos-do-Mar , Oligoelementos , Animais , Peso Molecular , Água do Mar , Análise Espectral/métodos , Oligoelementos/análise
6.
Int J Hematol ; 115(4): 481-488, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35088351

RESUMO

Hematopoietic stem cells (HSC) give rise to all types of blood lineages, including red blood cells (RBC). Hematopoietic stem/progenitor cells (HSPC) are known to be functionally diverse in terms of their self-renewal potential and lineage output. Consequently, investigation of molecular heterogeneity in the differentiation potential of HSPC is vital to identify novel regulators that affect generation of specific cell types, especially RBC. Here, we compared the erythroid potential of CD34+ hematopoietic stem and progenitor cells from 50 different umbilical cord blood (UCB) donors and discovered that those donors gave rise to diverse frequencies of Glycophorin-A+ erythroid cells after in vitro differentiation, despite having similar frequencies of phenotypic HSC initially. RNA sequencing revealed that genes involved in G protein-coupled receptor (GPCR) signaling were significantly up-regulated in the high-erythroid output donors. When we chemically modified two main signaling elements in this pathway, adenylyl cyclase (AC) and phosphodiesterase (PDE), we observed that inhibition of PDE led to 10 times higher yield of Glycophorin-A+ cells than activation of AC. Our findings suggest that GPCR signaling, and particularly the cAMP-related pathway, contributes to the diversity of erythroid potential among UCB donors.


Assuntos
Sangue Fetal , Transcriptoma , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Células-Tronco Hematopoéticas , Humanos
7.
Hum Cell ; 35(1): 408-417, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34817797

RESUMO

Ex vivo manufactured red blood cells (RBC) generated from immortalized erythroid cell lines which can continuously grow are expected to become a significant alternative in future transfusion therapies. The ectopic expression of human papilloma virus (HPV) E6/E7 gene has successfully been employed to establish these cell lines. To induce differentiation and maturation of the immortalized cell lines, terminating the HPV-E6/E7 expression through a gene induction system has been believed to be essential. Here, we report that erythroid cell lines established from human bone marrow using simple expression of HPV-E6/E7 are capable of normal erythroid differentiation, without turning gene expression off. Through simply changing cell culture conditions, a newly established cell line, Erythroid Line from Lund University (ELLU), is able to differentiate toward mature cells, including enucleated reticulocytes. ELLU is heterogeneous and, unexpectedly, clones expressing adult hemoglobin rapidly differentiate and produce fragile cells. Upon differentiation, other ELLU clones shift from fetal to adult hemoglobin expression, giving rise to more mature cells. Our findings propose that it is not necessary to employ gene induction systems to establish immortalized erythroid cell lines sustaining differentiation potential and describe novel cellular characteristics for desired functionally competent clones.


Assuntos
Diferenciação Celular , Células Eritroides , Expressão Gênica , Alphapapillomavirus/genética , Células da Medula Óssea , Linhagem Celular , Células Clonais , Genes Virais , Vetores Genéticos , Hemoglobinas , Humanos , Reticulócitos
8.
Molecules ; 26(18)2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34577173

RESUMO

In contrast to many human organs, only the human liver can self-regenerate, to some degree. Some marine echinoderms are convenient objects for studying the processes of regenerations of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix can completely restore within several weeks, the internal organs and the whole body after their division into two or three parts. Therefore, these cucumbers are a very convenient model for studying the general mechanisms of regeneration. However, there is no literature data yet on which biomolecules of these cucumbers can stimulate the regeneration of organs and the whole-body processes. Studying the mechanisms of restoration is very important for modern biology and medicine, since it can help researchers to understand which proteins, enzymes, hormones, or possible complexes can play an essential role in regeneration. This work is the first to analyze the possible content of very stable protein complexes in sea cucumbers Eupentacta fraudatrix. It has been shown that their organisms contain a very stable multiprotein complex of about 2000 kDa. This complex contains 15 proteins with molecular masses (MMs) >10 kDa and 21 small proteins and peptides with MMs 2.0-8.6 kDa. It is effectively destroyed only in the presence of 3.0 M MgCl2 and, to a lesser extent, 3.0 M NaCl, while the best dissociation occurs in the presence of 8.0 M urea + 0.1 M EDTA. Our data indicate that forming a very stable proteins complex occurs due to the combination of bridges formed by metal ions, electrostatic contacts, and hydrogen bonds.


Assuntos
Misturas Complexas/química , Proteínas/química , Pepinos-do-Mar/química , Animais , Ligação de Hidrogênio , Metais/química , Peso Molecular , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletricidade Estática , Engenharia Tecidual
9.
Commun Biol ; 4(1): 677, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083702

RESUMO

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.


Assuntos
Núcleo Celular/efeitos dos fármacos , Eritrócitos/metabolismo , Células Eritroides/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Inibidores de Histona Desacetilases/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Eritrócitos/citologia , Células Eritroides/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Histona Desacetilases/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Proc Natl Acad Sci U S A ; 117(35): 21267-21273, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817519

RESUMO

Introduction of exogenous genetic material into primary stem cells is essential for studying biological function and for clinical applications. Traditional delivery methods for nucleic acids, such as electroporation, have advanced the field, but have negative effects on stem cell function and viability. We introduce nanostraw-assisted transfection as an alternative method for RNA delivery to human hematopoietic stem and progenitor cells (HSPCs). Nanostraws are hollow alumina nanotubes that can be used to deliver biomolecules to living cells. We use nanostraws to target human primary HSPCs and show efficient delivery of mRNA, short interfering RNAs (siRNAs), DNA oligonucleotides, and dextrans of sizes ranging from 6 kDa to 2,000 kDa. Nanostraw-treated cells were fully functional and viable, with no impairment in their proliferative or colony-forming capacity, and showed similar long-term engraftment potential in vivo as untreated cells. Additionally, we found that gene expression of the cells was not perturbed by nanostraw treatment, while conventional electroporation changed the expression of more than 2,000 genes. Our results show that nanostraw-mediated transfection is a gentle alternative to established gene delivery methods, and uniquely suited for nonperturbative treatment of sensitive primary stem cells.


Assuntos
Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas , Nanoestruturas , Animais , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Microinjeções
11.
Blood Adv ; 4(9): 1833-1843, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32365188

RESUMO

Chemotherapeutic agents can reduce bone marrow (BM) activity, causing myelosuppression, a common life-threatening complication of cancer treatment. It is challenging to predict the patients in whom prolonged myelosuppression will occur, resulting in a delay or discontinuation of the treatment protocol. An early indicator of recovery from myelosuppression would thus be highly beneficial in clinical settings. In this study, bile acids (BAs) were highly increased in the systemic circulation as a natural response during recovery from myelosuppression, supporting regeneration of BM cells. BA levels in the blood of pediatric cancer patients and mice treated with chemotherapeutic agents were increased, in synchrony with early proliferation of BM cells and recovery from myelosuppression. In a mouse model of altered BA composition, Cyp8b1 knockout mice, a subset of mice recovered poorly after chemotherapy. The poor recovery correlated with low levels and changes in composition of BAs in the liver and systemic circulation. Conversely, BA supplementation in chemotherapy-treated wild-type mice resulted in significantly improved recovery. The results suggest that part of the mechanism by which BAs support recovery is the suppression of endoplasmic reticulum stress pathways in expanding and recovering hematopoietic cells. The findings propose a novel role of BAs as early markers of recovery and active components of the recovery process after chemotherapy.


Assuntos
Ácidos e Sais Biliares , Fígado , Animais , Medula Óssea , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout
12.
ScientificWorldJournal ; 2019: 9782635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31772512

RESUMO

In this paper, we have performed determination of the concentration of twenty elements in seven human organs (spleen, liver, kidney, muscle, heart, lungs, and brain) using two-jet plasma atomic emission spectrometry. The method allows multielemental analysis of solid samples without wet acid digestion. Before analysis, all human organs were first dried, ground to powders, and carbonized. The relative content of elements in each of the seven organs was very different depending on the donor. The average content of twenty elements in various organs varied in the following ranges (µg/g of dry weight): Ag (<0.02-0.2), Al (2.1-263), B (<0.5-2.5), Ca (323-1650), Cd (<0.1-114), Co (<0.2-1.0), Cr (<0.5-4.0), Cu (4.2-47), Fe (156-2900), Mg (603-1305), Mn (0.47-8.5), Mo (<0.2-4.9), Ni (<0.3-3.1), Pb (<0.3-1.9), Si (31.6-2390), Sn (<0.3-3.2), Sr (0.2-1.0), Ti (<2-31, mainly in lungs), and Zn (120-292). The concentration range of Ba in organs of five donors was <0.2-6.9 and 2.0-5600 for one donor with pneumoconiosis (baritosis). The maximum element contents were found, respectively, in the following organs: Al, B, Cr, Ni, Si, Sn, Sr, Ti (lungs), Fe (lungs and spleen), Mn (liver and kidney), Ag and Mo (liver), Ca (lungs and kidney), Cu (brain), Cd (kidney), Pb (brain), and Zn (liver, kidney, and muscle). The minimal content of elements was observed, respectively, in the following organs: Ag (all organs except liver), Ba (spleen, muscles, and brain), Ca and Mg (liver), Si (liver, muscle, and brain), Cd and Sr (heart and brain), Al, Cu, Fe, and Mn (muscle), and Zn (spleen and brain). The analysis of possible biological role and reasons for the increased content of some elements in the organs analyzed was carried out.


Assuntos
Análise Espectral , Oligoelementos/análise , Humanos , Especificidade de Órgãos , Análise Espectral/métodos , Oligoelementos/química
13.
ScientificWorldJournal ; 2019: 2578975, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467495

RESUMO

BACKGROUND: Many biological processes are performed by different protein complexes. During the association of proteins and enzymes forming specific complexes, the latter can include ions of various metal ions, which may be important for their formation and biological function. OBJECTIVE OF THE STUDIES: However, to date in the literature there are no data on metal ions that are part of any protein complexes. METHODS: A very stable multiprotein complex (~1000±100 kDa) was separated from other proteins of nine samples of female milk by gel filtration on Sepharose 4B. The content of microelements in the stable multiprotein complex and milk was analyzed using two-jet plasma atomic emission spectrometry. RESULTS: The content of different elements in milk on average decreased in the order: Ca>P>Mg>Al≥Zn≥Fe>Cu >B (0.76-3500 µg/g of dry milk powder), while the content of some elements was very low (Sr>Mn>Cr>Ba>Pb>Ag>Ni>Cd, <0.03-0.5 µg/g). The content of eight elements in stable multiprotein complex was 1.2-9.6-fold higher than in milk and increased in the order: Ca≈MgB (19.7)>Ag (28.7)>Ni (38)≥Sr (110). CONCLUSIONS: The analysis of the relative content of sixteen elements in human milk and oligomeric complexes of proteins was performed for the first time. Data on the content of metals indicate that during the formation of protein which associates the increase in the content of metal ions bound with proteins of the complex can occur. Such metal ions can be important for the formation and biological function of protein complexes.


Assuntos
Leite/química , Complexos Multiproteicos/metabolismo , Oligoelementos/análise , Animais , Feminino , Metais/análise , Peso Molecular , Espalhamento de Radiação , Fatores de Tempo
14.
Biomolecules ; 9(6)2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146486

RESUMO

Lactoferrin (LF) is a Fe3+-binding glycoprotein, that was first recognized in milk and then in other epithelial secretions and barrier body fluids to which many different functions have been attributed to LF including protection from iron-induced lipid peroxidation, immunomodulation, cell growth regulation, DNA and RNA binding, as well as transcriptional activation, еtс. The polyfunctional physiological role of LF is still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infections. It was shown previously that human milk LF possesses several enzymatic activities: DNase, RNase, ATPase, phosphatase, and amylase. Analysis of human, cow, horse, buffalo and camel LF showed a highly conserved three-dimensional (3D) structure including only detail differences in the species. Recently, it was shown that similar to human cow LF possesses DNase and RNase activities. Using different methods here we have shown for the first time that LFs from the milk of seven cows of different breeds possess high peroxidase, protease, amylase, protease, and phosphatase activities. Protease activity of cow LFs was activated by Mg2+ and Ca2+ ions. In contrast to human LFs, ATPase activity was revealed only in three of seven cow LF preparations. The discovery that LF possesses these activities may contribute to understanding the multiple physiological functions of this extremely polyfunctional protein including its protective role against microbial and viral infections.


Assuntos
Biocatálise , Lactoferrina/metabolismo , Leite/química , Animais , Bovinos , Hidrólise
15.
J Mol Recognit ; 32(7): e2777, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30761635

RESUMO

Lactoferrin (LF) is an Fe3+ -binding glycoprotein first recognized in milk and then in other epithelial secretions and barrier body fluids to which many different functions have been attributed to LF, including protection from iron-induced lipid peroxidation, immunomodulation, cell growth regulation, DNA and RNA binding, as well as transcriptional activation, еtс. The polyfunctional physiological role of LF is still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infections. Here, we present the first evidence that LF preparations isolated from milk of 18 cows of different breeds possess various levels of metal-dependent DNase and metal-independent RNase activities. For univocal assignment of DNase and RNase activities to cow LF, it was subjected to SDS-PAGE using gels with copolymerized calf thymus DNA or polymeric yeast RNA. In situ analysis was revealed DNase and RNase activities only in the gel zones corresponding to homogeneous LF. In contrast to human LF, cow LF possesses a relatively low cytotoxicity towards human tumor cells. The discovery that cow LF has these activities may contribute to understanding the multiple physiological functions of this extremely polyfunctional protein, including its protective role against microbial and viral infections. The computational spatial model of cow LF complex with DNA was obtained: according to the model positively charged residues of LF contact with DNA.


Assuntos
DNA/metabolismo , Lactoferrina/metabolismo , RNA Fúngico/metabolismo , Animais , Cruzamento , Bovinos , DNA/química , Feminino , Hidrólise , Lactoferrina/química , Modelos Moleculares , Conformação Proteica , RNA Fúngico/química , Timo/química , Leveduras/genética
16.
J Mol Recognit ; 31(12): e2753, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30109746

RESUMO

It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100 kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea + 1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95 kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95 kDa) but also few dozens of peptides with molecular masses from 2 to 9.5 kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Strongylocentrotus/embriologia , Animais , Cromatografia Líquida , Feminino , Peso Molecular , Óvulo/enzimologia , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Strongylocentrotus/enzimologia
17.
IUBMB Life ; 70(6): 501-510, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29601140

RESUMO

It is known that that human serum albumin (HSA) and alpha-lactalbumin (LA) possess DNA-binding sites. Electrophoretically homogeneous HSA and LA containing no canonical enzymes were isolated from human sera and milk. Here we have analyzed for the first time the possibility of DNA hydrolysis by these proteins. It was shown that HSA possesses metal-dependent DNase activity, while LA cannot hydrolyze DNA. Several rigid criteria have been applied to show that DNase activity is an intrinsic property of HSA from human sera and milk. HSA preparations were inactive after their dialysis against EDTA or in the presence of EDTA, but were activated after addition of several external metal ions: Mn2+ > Mg2+ > Ca2+ . The best activation of HSA preparations was observed in the presence of two metal ions: Mg2+ +Ca2+ > Mn2+ + Ca2+ ≥ Mn2+ + Mg2+ . In contrast to DNases having only one pH optimum, HSA preparations demonstrated two well-pronounced optima at pH 5.7-5.9 and 6.9-7.1 as well as a weak optimum at pH 8.4-8.6. These results demonstrate the diversity of HSA in the DNA hydrolysis at various pHs and in activation by various metal cofactors. Possible reasons for the diversity of HSA preparations are discussed. © 2018 IUBMB Life, 70(6):501-510, 2018.


Assuntos
DNA/metabolismo , Desoxirribonucleases/metabolismo , Metais/metabolismo , Leite Humano/metabolismo , Albumina Sérica Humana/metabolismo , Sítios de Ligação , Humanos , Hidrólise
18.
Biol Chem ; 399(4): 347-360, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29252186

RESUMO

We show here for the first time that HSA possesses two nucleic acid-(NA) binding sites and we estimated the relative contributions of the nucleotide links of (pN)n to their total affinity for these binding sites with higher and lower affinity for NAs. The minimal ligands of these binding sites are orthophosphate (Kd=3.0 and 20.0 mm), various dNMPs (5.6-400 µm and 0.063-18 mm) and different rNMPs (4.9-30 µm and 14-250 µm). Maximal contribution to the total affinity of all NAs to the first and second sites was observed for one nucleotide and was remarkably lower for three additional nucleotide units of (pN)n (n=1-4) with a significant decrease in the contribution at n=5-6, and at n≥7-8 all dependencies reached plateaus. For d(pA)n and r(pA)n a relatively gradual decrease in the contribution to the affinity at n=1-6 was observed, while several d(pN)n, demonstrated a sharp increase in the contribution at n=2-4. Finally, all (pN)n>10 demonstrated high affinity for the first (1.4-150 nm) and the second (80-2400 nm) sites of HSA. Double-stranded NAs showed significantly lower affinity comparing with single-stranded ligands. The thermodynamic parameters characterizing the specific contribution of every nucleotide link of all (pN)1-9 (ΔG°) to their total affinity for HSA were estimated.


Assuntos
DNA/metabolismo , RNA/metabolismo , Albumina Sérica Humana/metabolismo , DNA/química , Humanos , RNA/química , Albumina Sérica Humana/química , Termodinâmica
19.
Cell Stem Cell ; 18(4): 522-32, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26831518

RESUMO

During development, hematopoietic stem cells (HSCs) undergo a rapid expansion in the fetal liver (FL) before settling in the adult bone marrow. We recently reported that proliferating adult HSCs are vulnerable to ER stress caused by accumulation of mis-folded proteins. Here, we find that FL-HSCs, despite an increased protein synthesis rate and a requirement for protein folding, do not upregulate ER chaperones. Instead, bile acids (BAs), secreted from maternal and fetal liver, coordinate to serve as chemical chaperones. Taurocholic acid, the major BA in FL, supports growth of HSCs in vitro by inhibiting protein aggregation. In vivo, reducing BA levels leads to ER stress elevation and accumulation of aggregated proteins and significantly decreases the number of FL-HSCs. Taken together, these findings reveal that BA alleviation of ER stress is a mechanism required for HSC expansion during fetal hematopoiesis.


Assuntos
Ácidos e Sais Biliares/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Prenhez , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Retículo Endoplasmático/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Agregados Proteicos/efeitos dos fármacos
20.
J Mol Recognit ; 28(1): 20-34, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26268368

RESUMO

For breastfed infants, human milk is more than a source of nutrients; it furnishes a wide array of proteins, peptides, antibodies, and other components promoting neonatal growth and protecting infants from viral and bacterial infection. It has been proposed that most biological processes are performed by protein complexes. Therefore, identification and characterization of human milk components including protein complexes is important for understanding the function of milk. Using gel filtration, we have purified a stable high molecular mass (~1000 kDa) multiprotein complex (SPC) from 15 preparations of human milk. Light scattering and gel filtration showed that the SPC was stable in the presence of high concentrations of NaCl and MgCl2 but dissociated efficiently under the conditions that destroy immunocomplexes (2 M MgCl2 , 0.5 M NaCl, and 10 mM DTT). Such a stable complex is unlikely to be a casual associate of different proteins. The relative content of the individual SPCs varied from 6% to 25% of the total milk protein. According to electrophoretic and mass spectrometry analysis, all 15 SPCs contained lactoferrin (LF) and α-lactalbumin as major proteins, whereas human milk albumin and ß-casein were present in moderate or minor amounts; a different content of IgGs and sIgAs was observed. All SPCs efficiently hydrolyzed Plasmid supercoiled DNA and maltoheptaose. Some freshly prepared SPC preparations contained not only intact LF but also small amounts of its fragments, which appeared in all SPCs during their prolonged storage; the fragments, similar to intact LF, possessed DNase and amylase activities. LF is found in human epithelial secretions, barrier body fluids, and in the secondary granules of leukocytes. LF is a protein of the acute phase response and nonspecific defense against different types of microbial and viral infections. Therefore, LF complexes with other proteins may be important for its functions not only in human milk.


Assuntos
Amilases/metabolismo , Desoxirribonucleases/metabolismo , Leite Humano/química , Leite Humano/metabolismo , Complexos Multiproteicos/química , Amilases/química , Amilases/isolamento & purificação , Cromatografia em Gel , Desoxirribonucleases/química , Desoxirribonucleases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Peso Molecular , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Estabilidade Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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