UNLABELLED: Crayfish spermatophores are deposited on the body surface of the female during mating and remain there for a period of time before fertilization ensues. Post-mating changes in protein expression level in the noble crayfish Astacus astacus spermatophore were quantified. In-gel digestion and high resolution mass spectrometry were used for label-free protein quantification. One hundred twelve proteins were identified in the spermatophore of noble crayfish. After 7 days of storage on the body of the female, 6 proteins were identified in the post-mating spermatophore that showed significant up-regulation and 4 significant down-regulations (p < 0.05, fold change ≥ 2). The highest rate of up-regulation was observed in sodium/hydrogen exchanger, which may indicate the importance of intracellular pH adjustment for final maturation of the crayfish spermatozoon. The highest rate of down-regulation was observed in histone H2A. This may increase chromatin flexibility and facilitate its transfer into the oocyte during fertilization. The vitellogenin protein was identified in the crayfish spermatophore and its level changed during storage on the body surface of female. Extensive proteomic modification of male gametes during storage on the body surface of the female suggests post-mating final maturation of the crayfish spermatozoon. BIOLOGICAL SIGNIFICANCE: Freshwater crayfish comprise a large and diverse group of ecologically and commercially important animals. Molecular studies of gametes in the crayfish can provide insight into the complex process of reproduction in this diverse group of animals. The results of such studies can be used for development of new techniques for artificial reproduction of these economically important species.

• MeSH
• alfa-makroglobuliny metabolismus   MeSH
• chromatografie kapalinová MeSH
• hemokyanin metabolismus   MeSH
• histony metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kapacitace spermií MeSH
• Na(+)-H(+) antiport metabolismus   MeSH
• proteomika metody   MeSH
• ryanodinový receptor vápníkového kanálu metabolismus   MeSH
• severní raci fyziologie   MeSH
• sexuální faktory MeSH
• spermatogonie fyziologie   MeSH
• spermie fyziologie   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225-6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M (α2M-MA). Passage of high molecular weight α2M·hepcidin or α2M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M-MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells. Interestingly, α2M·hepcidin or α2M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2M or α2M-MA did not affect plasma clearance of α2M/α2M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α2M·hepcidin or α2M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2M or α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down-regulate Fpn1.

• MeSH
• alfa-makroglobuliny genetika   metabolismus   MeSH
• biologické modely * MeSH
• buněčné linie MeSH
• hepcidiny krev   genetika   MeSH
• lidé MeSH
• multiproteinové komplexy krev   genetika   MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• protein 1 související s LDL-receptory genetika   metabolismus   MeSH
• proteiny přenášející kationty biosyntéza   genetika   MeSH
• receptory LDL genetika   metabolismus   MeSH
• regulace genové exprese fyziologie   MeSH
• železo krev   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ovarian cancer is the most lethal of all gynecological cancers. Although serum biomarker CA125 is routinely used, there is a need for sensitive and specific complementary biomarkers. N-glycosylation changes in ovarian cancer serum glycoproteins include a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLex) on haptoglobin ß-chain, ?1-acid glycoprotein and ?1- antichymotrypsin. These changes are also present in chronic inflammations but not in malignant melanoma with low levels of inflammation. Acute phase proteins carrying increased amounts of SLex have an increased half-life. Sialylation of acute phase proteins decreases apoptosis favouring survival of cancer cells. Cancer cells produce inflammatory cytokines which influence glycosylation in liver parenchymal cells. The decreased galactosylation and sialylation of IgG increases cytotoxicity of natural killer cells and complement activation via mannosebinding lectin. Altered glycosylation of acute phase proteins and IgG suggests that cancer regulates certain pathways favouring survival of cancer cells.

• MeSH
• alfa-1-antichymotrypsin krev   metabolismus   MeSH
• alfa-makroglobuliny metabolismus   MeSH
• galaktosa nedostatek   MeSH
• glykosylace MeSH
• haptoglobiny metabolismus   MeSH
• imunoglobulin G krev   metabolismus   MeSH
• kyseliny sialové nedostatek   MeSH
• lidé MeSH
• metabolismus sacharidů MeSH
• nádorové biomarkery krev   MeSH
• nádory vaječníků imunologie   metabolismus   MeSH
• proteiny akutní fáze metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

• MeSH
• 2D gelová elektroforéza MeSH
• alfa-makroglobuliny metabolismus   MeSH
• gelová chromatografie MeSH
• kationické antimikrobiální peptidy metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• makrofágy cytologie   metabolismus   MeSH
• monocyty cytologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• proteiny přenášející kationty metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vazba proteinů MeSH
• western blotting MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• alfa-makroglobuliny fyziologie   klasifikace   metabolismus   MeSH
• ascitická tekutina patofyziologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• inhibitory syntézy proteinů klasifikace   metabolismus   MeSH
• tkáňové inhibitory metaloproteinas fyziologie   klasifikace   MeSH
• Publikační typ
• přehledy MeSH

• MeSH
• akutní myeloidní leukemie farmakoterapie   MeSH
• alfa-makroglobuliny metabolismus   MeSH
• dospělí MeSH
• hemostáza MeSH
• lidé MeSH
• plazminogen metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH