Age-Dependent Variation of Serum Protein Antigens in Sturgeons (Acipenseridae,Acipenseriformes)

We studied the variation of antigenic properties of proteins in two sturgeon species at different stages of postembryonic development. The deepest changes occurred in individual components of albumins and -globulins (transferrins) and were mostly related to an increased proportion of the protein accounting for these antigens. Transformation of the main component of albumins A1 into adult antigens was completed in 5-month old juveniles. The main component of -globulins A (component of transferrins) appeared in the blood flow much later than other proteins and could retain the juvenile features until the age of 3–4 years. Other antigens belonging to ₁- and ₂-globulins and the second component of transferrins were more stable and did not undergo substantial changes. The direction of ontogenetic variation of serum antigens in sturgeon juveniles did not depend on the habitat of adult fish in fresh or sea water.     

High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

High-resolution FISH mapping of the rat α2u-globulin multigene family

The rat α_2u-globulins are a group of similar proteins that are encoded by a family of approximately 20 genes located a single locus of ≤880 kbp on Chromosome (Chr) 5q. Individual members of this gene family demonstrate complex tissue, hormonal, and developmental expression patterns despite a high degree of sequence similarity among the members and consequently provide an interesting system for studying the evolution of differential gene expression. Hybridization analysis indicated that gene classes, similar to those identified at the homologous MUP locus in the mouse, do not exist within the rat α_2u-globulin locus. Furthermore, cross-hybridization analysis revealed the presence of conserved sequences in the 5′ and 3′ regions flanking the α_2u-globulin genes, some of which were present in an inverted orientation. We have used high-resolution fiber FISH to examine the structural organization of the α_2u-globulin locus, and found the genes to be arranged as an array of both direct and inverted repeats. The organization of the rat α_2u-globulin genes differs from the MUP genes and suggests different evolutionary events have reorganized these homologous sets of genes. Received: 26 July, 1999 / Accepted: 8 December 1999     

Genomic organization of the rat α2u-globulin gene cluster

The α_2u-globulins are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many α_2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 α_2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the α_2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the α_2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing α_2u-globulin genes indicated that the α_2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the α_2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes. Received: 12 August 1998 / Accepted: 13 January 1999     

Organization of diencephalon of the sturgeons. Dorsothalamic and epithalamic areas

Cytoarchitectonics of the dorsal part of diencephalon and epithalamus was studied in four species of the true sturgeons: the great sturgeon Huso huso L., the Kura sturgeon Acipenser gƅlden-st∂dtii persicus n. Kurensis Belyaeff, the starred sturgeon Ac. stellatus Pall., and the barbel sturgeon Ac. nudiventris Lov. Material was processed using routine Nissl and Bielschowski staining techniques (the latter in Viktorov’s modification). The described areas are the smallest among diencephalic regions. Dorsal thalamus is exceeded by the ventral one both in length and in the number nuclear structures. It includes four nuclear structures, three of which are periventricular: the anterior, posterior dorsal, and central nuclei, while the subhabenular nucleus is a migrated one. Epithalamic area has a pronounced right-left asymmetry that consists both in the external appearance of these structures and in their internal organization. Three types of neurons were revealed in epithalamic structures: granular cells, rounded neurons with poorly expressed processes, and bipolar neurons with clearly seen processes. In the epithalamic area, a sufficiently large nervous structure designated as the central habenular nucleus consisting of relatively large rounded neurons was described. A conclusion is made about a unique organization the studied areas in the true sturgeons, which distinguishes them markedly among both other actinopterygian fish and the higher organized vertebrates.     

Spectrophotometric parameters of conformational states of hemoglobin and its complexes with ligands in the sturgeons

Parameters of carbo-, carboxy-, oxy-, deoxy-, and methemoglobin of the human and four species of sturgeons (sterlet, Russian sturgeon, starred sturgeon, great sturgeon) were analyzed spectrophotometrically. It has been shown that in the absorption spectra of all forms of hemoglobins there is only one Soret γ₁ band due to the prosthetic group of the pigment; the wavelength of this band is undoubtedly associated with the hemoglobin conformation. This permits the use of the band as an indicator parameter to control an initial state of the fish hemoglobin solutions, the analysis of conformational states of the pigment macromolecules, and the study of hemoglobin derivatives (its complexes with ligands).     

Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio)

A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α₁-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of α₁-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of α₁-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum α₁-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically. Received June 4, 1998; accepted September 10, 1998.     

Spectrophotometric parameters of conformational states of hemoglobin and its complexes with ligands in the sturgeons

Parameters of carbo-, carboxy-, oxy-, deoxy-, and methemoglobin of the human and four species of sturgeons (sterlet, Russian sturgeon, starred sturgeon, great sturgeon) were analyzed spectrophotometrically. It has been shown that in the absorption spectra of all forms of hemoglobins there is only one Soret γ₁ band due to the prosthetic group of the pigment; the wavelength of this band is undoubtedly associated with the hemoglobin conformation. This permits the use of the band as an indicator parameter to control an initial state of the fish hemoglobin solutions, the analysis of conformational states of the pigment macromolecules, and the study of hemoglobin derivatives (its complexes with ligands).     

Inheritance of some quantitative morphological characters in reciprocal hybrids of starred sturgeon <Emphasis Type="Italic">Acipenser stellatus</Emphasis>and beluga <Emphasis Type="Italic">A. huso</Emphasis> (Acipenseridae)

Some quantitative characters of three-summer old reciprocal hybrids of beluga Acipenser huso and starred sturgeon A. stellatus,as well as of juveniles of their parental species, are comparatively analyzed. The found maternal and paternal effects in manifestation of some characteristics in hybrids are considered in connection with elucidation of inheritance type of morphological characters in acipenserids. The paternal effect observed by some characters in hybrids beluga × starred sturgeon is explained by prevalence of dominant alleles in the genome of starred sturgeon in comparison with beluga. The maternal effect is elucidated by a few characteristics related to parameters of the head, first of all to the volume of the brain region of skull. Evidently, in this case, the matrocliny depends on the fact that information on the number of segments of embryo and their position is determined by the maternal organism during oogenesis.     

Age-related variability of serum protein antigens in sturgeons (Acipenseridae, Acipenseriformes)

We studied the variability of antigenic properties of proteins in two sturgeon species at different stages of postembryonic development. The deepest changes occurred in individual components of albumins and beta-globulins (transferrins) and were mostly related to an increased proportion of the protein accounting for these antigens. Transformation of the main component of albumins A1 into adult antigens was completed in 5-month old fry. The main component of beta-globulins A (component of transferrins) appeared in the blood flow much later than other proteins and could retain the fry features until the age of 3-4 years. Other antigens belonging to alpha1- and alpha2-globulins and the second component of transferrins were more stable and did not undergo substantial changes. The direction of ontogenetic variability of serum antigens in sturgeon fry did not depend on the habitat of adult fish in fresh or sea water.     

Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.     

Structural and functional characterization of <Emphasis Type="Italic">Delphinus delphis</Emphasis> hemoglobin system

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC–electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major β (β1 16,022 Da, β2 16,036 Da, β3 16,036 Da, labeled according to their progressive elution times) and two major α globins (α1 15,345 Da, α2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that β2 globin differs from β1 for the substitution Val126 → Leu, while β3 globin differs from β2 for the isobaric substitution Lys65 → Gln. The α2 globin differs from the α1 for the substitution Ser15 → Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC–ESI-MS analysis revealed that the fraction with higher pI (HbI) contained β1, β2 and both the α globins, and the fraction with lower pI (HbII) contained β3 and both the α globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA₀, with HbII showing the more pronounced differences. With respect to HbA₀, either the substitution Proβ5 → Gly or the Proβ5 → Ala is present in all the cetacean β globins sequenced so far, and it has been hypothesized that position 5 of β globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysβ65 → Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA₀.     

Comparative enzymologic study of catalytical properties of liver monoamine oxidases of sturgeon fish

We carried out the comparative study of the substrate and inhibitory specificity of liver monoamine oxidases (MAO) of the giant sturgeon Huso huso, the starred sturgeon Acipenser stellatus, the Persian sturgeon Acipenser persicus, and the Russian sturgeon Acipenser gueldenstaedtii. Results of the substrate-inhibitor analysis with use of inhibitors chlorgilin and deprenil, as well as five specific substrates indicate homogeneity of these enzymes. All studied MAO have the several orders higher sensitivity to chlorgilin than to deprenil, with essential interspecies differences being observed. There are determined kinetic parameters of enzymatic deamination (K _M and V) of tyramine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, and N-methylhistamine. All studied enzymes have been established to have the higher activity toward serotonin and noradrenalin-substrates of the MAO A form as compared with benzylamine, β-phenylethylamine, and N-methylhistamine-substrate of the mammalian MAO B form, the maximal activity being characteristic of the giant sturgeon.     

Immunochemical study of the specific antigens of human amniotic fluid]

Four specific antigens (trophoblastic beta 1-globulin, placental lactogen, alpha 1- and alpha 2-globulins of human placenta) were identified using antisera to the native amniotic fluid. Five antigens with the mobility of prealbumins, alpha 1-globulins, alpha 2-globulins and beta 2-globulins which bear no resemblance with the previously studied antigens were identified using antisera to the acid fraction of the amniotic fluid. Both the prealbumins and alpha 2-globulin were found in the blood serum of foetuses of different age and of newborn infants; these proteins were absent from the blood serum of pregnant women and donors. They received the names of embryonic prealbumine 1, embryonic prealbumine 2 and embryonic alpha 2-globulin. The protein with the mobility of alpha 1-globulins was found in the amniotic fluid of foetuses and in the blood serum of pregnant women only and received the name of amniotic alpha 1-globulin. The concentration of the antigens in question was studied in the developing foetuses and in the blood serum of pregnant women at different stages of pregnancy.     

Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines

Recent studies have revealed an unexpected synergism between two seemingly unrelated protein families: CCN matricellular proteins and the tumor necrosis factor (TNF) family of cytokines. CCN proteins are dynamically expressed at sites of injury repair and inflammation, where TNF cytokines are also expressed. Although TNFα is an apoptotic inducer in some cancer cells, it activates NFκB to promote survival and proliferation in normal cells, and its cytotoxicity requires inhibition of de novo protein synthesis or NFκB signaling. The presence of CCN1, CCN2, or CCN3 overrides this requirement and unmasks the apoptotic potential of TNFα, thus converting TNFα from a proliferation-promoting protein into an apoptotic inducer. These CCN proteins also enhance the cytotoxicity of other TNF cytokines, including LTα, FasL, and TRAIL. Mechanistically, CCNs function through integrin α₆β₁ and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation, which is essential for apoptotic synergism. Mutant CCN1 proteins defective for binding α₆β₁-HSPGs are unable to induce ROS or apoptotic synergism with TNF cytokines. Further, knockin mice that express an α₆β₁-HSPG-binding defective CCN1 are blunted in TNFα- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. These findings implicate CCN proteins as contextual regulators of the inflammatory response by dictating or enhancing the cytotoxicity of TNFα and related cytokines.     

Variability of some blood serum β-globulins in life cycle of sturgeons (Acipenseridae)

Blood proteins of sturgeons of two genera Acipenser and Pseudoscaphirhynchus preserving unidirectional variability in ontogenesis regardless of peculiarities of the fish ecology have heterochronic character. The slowest ontogenetic development lasting for 4–6 years is peculiar to one of antigens of β-globulins. Analysis of the variability of β-globulins in the age range beginning from the 4-month juveniles and including adult spawned individuals indicates different functional significance of their individual components. The βA-globulin absent at early postembryonic stages becomes the second, after albumin, quantitatively predominant antigen in the adult fish blood. The amount of βB-globulin, like antigens of α₁and α₂-globulins preserving relative ontogenetic stability, can decrease significantly in freshwater sturgeons due to their preparation for spawning.     

Genes for two homologous G-protein α subunits map to different human chromosomes

Summary Signal transduction across biological membranes is modulated by a family of related GTP-binding proteins termed G proteins. These G proteins have a heterotrimeric structure composed of α, β, and γ subunits. The α subunits of the G proteins bind GTP and appear to determine the biochemical specificity of the protein. We have recently cloned and characterized cDNA encoding two G-protein α subunits, α_i and α_h. The former is a substrate for ADP-ribosylation by pertussis toxin. The protein corresponding to α_h has not yet been identified. These cDNAs encode proteins, which demonstrate 90% sequence identity to one another and also show marked similarity to other G proteins. The present studies were designed to determine whether the genes for these related proteins are clustered on a single human chromosome. Genomic DNA isolated from a panel of mouse-human hybrid cell lines was analyzed by hybridization to cDNAs for α_i and α_h. Based on the distribution patterns of α_i and α_h in cell hybrids, the gene for α_i was assigned to human chromosome 7, and the gene for α_h assigned to chromosome 12. These data suggest that the G-protein gene family may be distributed over at least two human chromosomes.     

Factors influencing the natural reproduction decline in the beluga (Huso huso,Linnaeus, 1758), Russian sturgeon (Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833), and stellate sturgeon (A. stellatus,Pallas, 1771) of the Volga–Caspian basin: A review

The review describes the changes in natural reproduction of three important sturgeon species in the Volga–Caspian basin: (a) the beluga (Huso huso Linneaus, 1758), (b) the Russian sturgeon (Acipenser gueldenstaedtii Brandt &Ratzeburg, 1833), and (c) the stellate sturgeon (Acipenser stellatus Pallas, 1771). Since the past 60 years, these species responded to severe influences of natural and anthropogenic factors. On the basis of original and published data, an analysis has been made of (a) the numbers of larvae migrating from spawning sites (according to plankton net survey), (b) fecundity and histological anomalies in gonad development, (c) the numbers of adult sturgeons in the Caspian Sea and of spawners migrating to the Volga River (according to trawl and beach seine survey), and (d) foraging resources for the sturgeons. The results show that their natural reproduction in the Volga–Caspian basin has declined drastically during the past decades under the impact of (a) fluctuations of the Caspian Sea level and flow discharge from the Volga River, (b) blockage of sturgeon migration routes and loss of spawning sites because of dam construction, (c) water pollution in the lower reaches of the Volga River and in the Caspian Sea, and (d) intensive and selective illegal and unreported fishing. The relative significance of these factors has been changing during the study period.     

Delayed internalization and lack of recycling in a beta<Subscript>2</Subscript>-adrenergic receptor fused to the G protein alpha-subunit

Background  

Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β₂-adrenergic receptor (β₂AR) and Gα_s indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gα_s-fused β₂AR.     

Multiple G<Subscript>i</Subscript> Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G_i/o proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Gα_z or the PTX-resistant mutants of Gα_i1–3 and Gα_oA. Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G_i1–3 proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.