Oligonucleotide uptake in leukocytes is dependent on extracellular calcium: a hint for the involvement of adhesion molecules?

Abstract

An enhanced appreciation of internalization of antisense oligonucleotides will extend possible applications in experimental and therapeutic settings. We found that oligonucleotide incorporation in monocytes, granulocytes and B-lymphocytes but not in T-lymphocytes is strongly dependent on the presence of extracellular calcium, and is inhibited by heparin. Our results support the hypothesis that calcium-dependent adhesion molecules mediate oligonucleotide internalization in leukocytes.     

Modeling control of Common Carp (Cyprinus carpio) in a shallow lake–wetland system

Wetlands Ecology and Management - The introduction of Common Carp (Cyprinus carpio) into North American waterways has led to widespread alteration of aquatic ecosystems. Control of this invader has...     

Adaptation of fatty acid composition to temperature—a study on carp (Cyprinus carpio L.) liver slices

1. Liver slices obtained from warm-, and cold-adapted carp were incubated in the presence of [1-¹⁴C]sodium acetate, -stearate, -linoleate, and -linolenate at various temperatures and the distribution of radioactivity among different phospholipid fatty acids was determined.2. Relative labelling of saturated fatty acids is reduced, while that of long chain polyunsaturated fatty acids, especially of docosahexanoate, is increased with decreasing temperatures.3. Liver slices of cold adapted carp produced a fatty acid population at 25°C indistinguishable from that produced by warm adapted ones at the same temperature.4. Liver slices obtained from cold-, and warm-adapted animals start to reorganise the pattern of labelling immediately after the exposure to the opposite temperatures as evident from pulse-chase labelling experiments.5. Desaturation of saturated and various unsaturated fatty acids is initiated immediately after down-shift of the temperature. This cold induced increase in desaturase activity is prevented by cycloheximide in the incubation medium.6. It is concluded that phospholipid fatty acid composition is continuously adjusted to the temperature and is governed partly by temperature coefficient of fatty acid synthetase and partly by induction or deactivation of desaturases in cold and warm, respectively.     

Genetic polymorphism of lactate dehydrogenase isoenzymes in the carp (Cyprinus carpio) apparently due to a “null allele”

As a consequence of polyploidization, the carp is endowed with three genetic loci coding for LDH in most tissues (and additional ones in liver). However, each such duplicated gene may not be functionally essential for the organism and could be eliminated. In a population survey, an electrophoretic polymorphism was detected which may best be interpreted by the assumption of a null allele, which is apparently not subject to selective pressure. Thus this originally duplicated gene would have diverged in the course of evolution without the origin of differences in function, so that one or the other of the genes is dispensable.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Are beta-adrenergic receptors in ventricular muscles of carp heart (Cyprinus carpio) mostly the beta-2 type?

The positive inotropic effect of isoproterenol was quantified in the presence of several beta-adrenergic blocking agents in ventricular strips of carp heart. Isoproterenol had a concentration-dependent positive inotropic effect. The effect was markedly inhibited by propranolol and carteolol, but was extremely insensitive to atenolol. Practolol totally failed to alter the effect. These results indicated that the positive inotropic effect of isoproterenol may be mediated by mostly beta-2 adrenergic receptors in ventricular strips of carp heart.     

Effect of electrical stimulation on β-adrenergic receptor population and cyclic AMP production in chicken and rat skeletal muscle cell cultures

Summary Expression of the β-adrenergic receptor (βAR) and its coupling to cyclic AMP (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the βAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically, chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7d in culture were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the βAR population was not significantly affected by electrical stimulation; however, the ability of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the βAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.     

The effect of prostaglandins on ox pituitary content of adenosine 3′:5′-cyclic monophosphate and the release of growth hormone

1. An assay, based on competition between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and cyclic [(3)H]AMP for binding to a rabbit skeletal muscle protein, has been used to measure tissue contents of cyclic AMP. The assay has a sensitivity of 0.05pmol of cyclic AMP. Cyclic GMP and cyclic CMP have 0.5%, and cyclic IMP 6.5%, of the ability of cyclic AMP to displace cyclic [(3)H]AMP from binding protein; AMP, ADP and ATP have no effect. 2. By using this method, the cyclic AMP content of ox pituitary slices exposed to prostaglandin was determined; release of growth hormone was measured by radioimmunoassay. 3. Release of growth hormone was increased by 45min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 10nm-prostaglandin E(2), 0.1mum-prostaglandin A(1) or 1mum-prostaglandin B(1) in the presence of 0.5mm-theophylline. 4. Pituitary cyclic AMP content was increased by 10min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 0.1mum-prostaglandin E(2) in the presence of 0.5mm-theophylline. 5. The maximum increase in cyclic AMP content was observed 10min, and significant changes in growth hormone release 30min, after introduction of prostaglandin E(2). 6. The increase in pituitary cyclic AMP content, but not in the rate of release of growth hormone, was observed in the absence of external Ca(2+). 7. The stimulation of release of growth hormone by prostaglandin was decreased by preincubation of tissue for 2h in colchicine (100mum) or cytochalasin B (10mug/ml). 8. These results support the suggestion that increased release of growth hormone after treatment with prostaglandin is the result of increased tissue cyclic AMP content, and possibly involves a microfilamentous or microtubular protein.     

Formation of guanosine 3′,5′-cyclic monophosphate by thyroliberlin in cultured rat pituitary cells a possible role in stimulation of prolactin synthesis

In a clonal strain of rat pituitary tumour cells (GH₄C₁ cells), thyroliberin stimulated prolactin secretion and synthesis: effects that could be demonstrated after 5 min and 4–5 h of treatment, respectively. Within 0.5–5 min after addition of thyroliberin, maximal increases (2–4 hold) in cellular cyclic GMP concentrations were observed, and this rise preceded or occurred simultaneously with that of cyclic AMP. After 60 min of treatment the concentrations of the cyclic nucleotides had returned to control values. Half maximal and maximal stimulation of cyclic GMP elevations were obtained with approx. 2·10⁹ and approx. 27·10^?9 thyroliberin, respectively. Aminophylline increased both cyclic GMP and cyclic AMP, and potentiated the stimulatory effects of thyroliberin on both cyclic nucleotides. The dibutyryl derivative of cyclic GMP (10^?4–10^?6 M) stimulated prolactin synthesis, but not hormone release. Prostaglandin E₂ (3·10^?7 M) stimulated cellular cyclic AMP concentrations, but did not affect cyclic GMP levels. We conclude that thyroliberin in the GH₄C₁ ccell strain stimulates cyclic GMP formation, in addition to elevate cyclic AMP concentrations. The stimulatory effect on cyclic GMP is probably not secondary to the rise in cyclic AMP concentration, since prostaglandin E₂ elevates only cyclic GMP is involved in the action of thyroliberin on prolactin, the present results suggest a role on hormone synthesis.     

Oligomerization of 3′-amino-3′-deoxyguanosine-5′-phosphorimidazolidate on a d(CpCpCpCpC) template

Summary 3-Amino-3-deoxyguanosine-5-phosphorimidazolidate (ImpGnh ₂) oligomerizes more rapidly and regiospecifically than related nucleotide derivatives on a d(CpCpCpCpC) template. The greater nucleophilicity of the amino group leads to efficient oligomerization even when the structure of the double-helical complex formed by the template and the substrate is not optimal for reaction. The use of amine-containing analogues should permit us to develop models of potentially prebiotic polymerization reactions that cannot be studied easily using natural nucleotides.     

Cloning and expression analysis in mature individuals of two chicken type-Ⅱ GnRH (cGnRH-Ⅱ) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH), the pivotal signal molecular of hypothalamic-pituitary- gonad (HPG) axis, plays a crucial role in gonadal de-velopment and maintenance of reproduction function of vertebrates by stimulating the anterior pituitary re-leasing gonadotropin (GtH). Mammal GnRH (mGn- RH) was firstly identified from porcine and ovine in the 1970s[1,2], which was originally named luteinizing hormone releasing hormone, viz LHRH. To date, 16 GnRH variants have been characte…     

Effects of epoxymethano analogues of prostaglandin endoperoxides on aggregation,on release of 5-hydroxytryptamine and on the metabolism of 3′,5′-cyclic AMP and cyclic GMP in human platelets

The effects on human platelets of two synthetic analogues of prostaglandin endoperoxides were examined in order to explore the relationship between aggregation and prostaglandin and cyclic nucleotide metabolism, and to help elucidate the role of the natural endoperoxide intermediates in regulating platelet function.Both analogues (Compound I, (15S)-hydroxy-9α,11α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid, and Compound II, (15S)-hydroxy-11α,9α-(epoxymethano)-prosta-(5Z,13E)-dienoic acid) caused platelets to aggregate, an effect which could be inhibited by prostaglandin E₁ but not by indomethacin. Compound II produced primary, reversible aggregation at concentrations which did not induce release of 5-hydroxytryptamine. Production of thromboxane B₂ and malonyldialdehyde was monitored as an index of endogenous production of prostaglandin endoperoxides and thromboxane A₂ and were increased after incubation of human platelets with thrombin, collagen or arachidonic acid. However, neither malonydialdehyde nor thromboxane B₂ levels were significantly influenced by the endoperoxide analogues. Both analogues produced a small elevation of adenylate cyclase activity in platelet membranes and of cyclic AMP content in intact platelets, but neither had any modifying effect on the much greater stimulation of adenylate cyclase and cyclic AMP levels by prostaglandin E₁. Of all the aggregating agents tested, only arachidonic acid produced any significant increase in platelet cyclic GMP levels.These results suggest that the epoxymethano analogues of prostaglandin endoperoxides induce platelet aggregation independently of thromboxane biosynthesis and without inhibiting adenylate cyclase or lowerin platelet cyclic AMP levels. They therefore differ from better known aggregating agents such as ADP, epinephrine and collagen, which increase thromboxane A₂ production and reduce cyclic AMP levels, at least in platelets previously exposed to prostaglandin E₁.     

Multiple forms of α2-macroglobulin from a bony fish, the common carp (Cyprinus carpio): striking sequence diversity in functional sites

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the alpha2-macroglobulin (alpha2M) protein family, presumably expanding the diversity of immune recognition. To examine whether the alpha2M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of alpha2M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct alpha2M sequences from a single individual, indicating the presence of multiple alpha2M genes in this species. The deduced amino acid sequences contained a post-translational cleavage signal, predicting a C3-like two-chain structure, as in lamprey alpha2M. Two distinct alpha2M proteins were purified from carp serum; both proved to be Mr 380,000 dimers, the subunits of which are composed of disulfide-linked alpha chains (Mr 93,000) and beta chains (Mr 85,000), as reported for the alpha2M from plaice, another teleost species. The presence of an internal thioester in the alpha chain was demonstrated by its autolytic fragmentation and direct incorporation of [14C]methylamine. Interestingly, the three forms of carp alpha2M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the alpha chain assigned as the receptor-binding domain, while an Asn residue at the position corresponding to the catalytic His in C3 was completely conserved in the carp alpha2Ms, as in most alpha2Ms of other animals. The possible functional significance of the sequence diversity is discussed.     

Effect of dibutyryl cyclic adenosine 3′5′-monophosphate on mitotic activity in the thyroid of hypophysectomized rats

Summary Effect of dibutyryl cyclic adenosine 35-monophosphate (dbcAMP) on mitotic activity in the thyroid of hypophysectomized rats has been examined. It has been demonstrated that dbcAMP stimulates the incidence of mitoses in the thyroid follicular cells. It is therefore suggested that cAMP may be a mediator of the proliferogenic effect of TSH on the thyroid in vivo. Cyclic AMP could also release some unidentified growth-promoting factors for the thyroid. A direct stimulating effect of dbcAMP on the proliferation of the thyroid follicular cells is assumed to be possible as well.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Characterization and expression pattern of a novel β-defensin in common carp (Cyprinus carpio L.): implications for its role in mucosal immunity

β-defensins are a group of cysteine-rich cationic antimicrobial peptides that play antibacterial and antiviral roles in immune systems of vertebrates. Here, we report the cloning and identification of a β-defensin 3 cDNA sequence from the common carp (Cyprinus carpio L.). Sequence alignment and phylogenetic analysis indicated that this β-defensin 3 belonged to the BD-2 group of fish. Real-time PCR showed that the β-defensin 3 mRNA was expressed in all the tissues of normal common carp that we examined and was highly expressed in the spleen and gills. When challenged with Vibrio anguillarum, the expression level of common carp β-defensin 3 mRNA was quickly upregulated in various tissues. Our results indicate that the β-defensin 3 showed markedly high constitutive expression in the gills, and significantly upregulated expression in the hindgut of the common carp after infection, suggesting it plays an important role in the innate and mucosal immunity of common carp.