Ancient Cytokines,the Role of Astakines as Hematopoietic Growth Factors

Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.     

Interpretation of invertebrate photoreceptor potentials in terms of a quantitative model

It is known from voltage-clamp experiments on visual cells of Limulus and Balanus that the total membrane current can be separated approximately into a dark current J _D and a light-induced current J _L such that each part has a time-and intensity-independent reversal potential. In addition J _L can be represented approximately as a product of a nonlinear, time-independent current-voltage characteristics J ⁰ _L (V) and an activation factor x _A which depends on light intensity and time. J ⁰ _L (V) can be described by a simple electrodiffusion membrane model (for J _D we use the same model). A set of kinetic equations including amplification, latency and light adaptation leads to a determination of x _A for photoisomerization of single rhodospin molecules and for arbitrary light signals. The receptor potentials calculated show many features of the experiments on Limulus, Balanus and Astacus.List of the more Important Symbols A Adaptation factor - C ratio between slopes of current-voltage curves for maximum light-induced current and dark current - d one half the thickness of the membrane - F _i effective membrane permeability, cf. Eq. (7) - g _i electrochemical potential inside membrane - g _i electrochemical potential inside and outside cell - I light intensity (quanta/sec cm²) - i (index) refers to i-th ionic species - J total current leaving cell - J _D dark current - J _L light-induced current - j _i ionic current density inside membrane in x-direction - K cf. Eq. (21) - k Boltzmann's constant - k ₀ rate constant for chain reaction (production of X) - k ₁ rate constant for decay of X ₁ - k ₂ rate constant for decay of X (closing of sites) - M total number of sites controlling light conductance - N multiplication factor in dark adapted state (maximum of Z) - n _i density of ions iaside membrane - n _i density of ions inside and outside cell - p ₁, p ₂ rate constants for adaptation kinetics - p ₃ cf. Eq. (29) - q elementary charge - q _i ionic charges - r = (x, y, z) spatial variable - T temperature - t time - u cf. Eq. (28) - V membrane potential - V _D reversal potential for the dark current - V _L reversal potential for the light current - V _i diffusion potential, cf. Eq. (8) - V potential steps at the inner and outer membrane surface - V ₀ = V ^–V - X = x _a M, number of open sites - X ₁ = x ₁M number of molecules of agent whose decay initiates chain reactions - x _A degree of activation of light sensitive membrane - Y number of sites opened by individual chain reaction - Z multiplication factor (number of sites opened by one photoisomerized rhodopsin molecule) - _i activation energy inside membrane (measured with respect to surrounding solution) - electrostatic potential - ₀ flxed-charge distribution inside membrane - cross section for photoisomerization of one rhodopsin molecule by a photon - k ₁/k ₂     

Quantitative model for the electric response of invertebrate and vertebrate photoreceptors

We propose that the same mechanism which leads to light-adaptation in invertebrate photoreceptors is responsible for the excitation of the receptor potential in vertebrates. Several qualitative and quantitative features of the vertebrate receptor response support this hypothesis.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, October 4–8, 1976     

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.     

Changes in cytokine production and impaired hematopoiesis in patients with anorexia nervosa: the effect of refeeding

The changes in cytokines and hormones involved in hematopoiesis were studied in the serum of 7 girls with anorexia nervosa, 15-24 yr old, on admission and after 5% and 10% weight gain. Hematopoiesis was studied by in-vitro culturing of circulating granulocyte-macrophage colony forming cells and erythroid burst forming cells. Nutritional status was studied by anthropometric measurements and resting energy expenditure. On admission, granulocyte-macrophage colony forming cells and erythroid burst forming cells were significantly lower than in age-matched controls and increased significantly along weight gain. Blood leptin and erythropoietin levels increased significantly with weight gain. TNF-alpha levels tended to decrease while IL-1beta levels were lower than in the controls on admission (p <0.05) and did not change significantly during weight gain. IL-3, GM-CSF and IL-6 were undetected on admission or along weight gain. The changes in granulocyte-macrophage colony forming cells and erythroid burst forming cells positively correlated with changes in resting energy expenditure and fat free mass. These results may suggest that undernutrition affects hematopoiesis as indicated by the reduction of hematopoietic progenitor cells before treatment and the significant increase with weight gain. The changes in the levels of hormones and cytokines known to be involved in hematopoiesis along refeeding may suggest a role for these factors in anorexia nervosa.     

Structure and function of invertebrate 5-HT receptors: a review

Over the last decade, knowledge of invertebrate serotonin receptors has expanded greatly. The first 5-HT receptor from Drosophila was cloned 10 years ago, and subsequently, 11 additional receptor genes have been cloned from Drosophila, molluscs (Lymnaea and Aplysia) and nematodes (Caenorhabditis and Ascaris). Information has also accumulated from physiological and biochemical studies that have used vertebrate serotonergic ligands to characterize endogenous invertebrate receptors. Although the endogenous receptors are often classified according to mammalian-based categories, in many cases the pharmacological properties of vertebrate and invertebrate receptors differ significantly and the actual identity of the latter is questionable. By providing information on the gene structure and amino acid sequence, molecular cloning studies offer a more definitive way to identify and classify invertebrate 5-HT receptors. This review summarizes information on the pharmacological and transductional properties of cloned invertebrate 5-HT receptors, and considers recent studies of endogenous receptors in the light of this new data.     

Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity

Hematopoiesis is the process by which blood cells (hemocytes) mature and subsequently enter the circulation and we have developed a new technique to culture the hematopoietic progenitor cells in vitro. The reason for the successful culture was the isolation of a plasma protein that turned out to be a novel cytokine, astakine 1 (Ast1) containing a domain present in several vertebrates, so-called prokineticins. Now we have detected several astakines from other invertebrate species. Depending on our discovery of the cytokine Ast1 we have an opportunity to study in detail the differentiation of cells in the hematopoietic tissue of a crustacean, a tissue of evolutionary interest for studies of the connection between the vascular system and the nervous system. We have been able to isolate the entire hematopoietic tissue and for the first time detected a link between this tissue and the brain. We have further localized a proliferation center in the tissue and characterized its different parts. We have also used this system to isolate a new hematopoietic factor CHF that is important in the crossroad between apoptosis and hemocyte differentiation. Our technique for culture of crayfish hematopoietic stem cells provides a simple tool for studying the mechanism of hematopoiesis, but also enables detailed studies of immune defense reactions. Further, the culture system has been used for studies of viral defense and the system is suitable for gene silencing which allows functional characterization of different molecules involved in host defense as well as in hemocyte differentiation.     

Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation.     

Identification of a gp130 cytokine receptor critical site involved in oncostatin M response

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.     

Cloning of a cDNA that encodes an invertebrate glutamate receptor subunit.

The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N-terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N-terininal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N-terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N-terminal end of fibronectin, may be structurally related.     

Identification of genes involved in growth inhibition of breast cancer cells transduced with estrogen receptor

Estrogen receptor alpha (ERalpha)-negative breast cancer cells display an aggressive phenotype. We previously showed that adenoviral expression of ERalpha in ER-negative breast cancer cells leads to an estrogen-dependent down-regulation of the proliferation, which could be of interest to control the growth of such cells. In this study, we observed an increase in protein levels of p21 and p27 cyclin-dependent kinase inhibitors, whereas pRb phosphorylation is strongly decreased. Flow cytometry experiments showed a slower transit of cells in G1 (hormone-independent), a hormone-induced accelerated transit through S phase and a possible arrest in G2/M phase. In addition, ERalpha-expressing cells were undergoing apoptosis. By using cDNA macroarrays, we identified a novel collection of genes regulated by liganded ERalpha potentially regulating cell cycle, apoptosis, cell signalling, stress response and DNA repair.     

Structural similarities between a mitochondrially encoded polypeptide and a family of prokaryotic respiratory toxins involved in plasmid maintenance suggest a novel mechanism for the evolutionary maintenance of mitochondrial DNA

Summary Subunit 8 of mitochondrial ATP synthase (A8), a mitochondrially encoded polypeptide, has no known homologue in any prokaryotic or plastid ATP synthase, suggesting that it has been recruited to its present role in the enzyme from an extraneous source. The polypeptide is poorly conserved at the primary sequence level, but shows a well-conserved hydropathy profile. The hydropathy profiles of A8 from diverse taxa were compared with those of thehok family of prokaryotic respiratory toxins, some of whose members are involved in plasmid maintenance, through postsegregational killing of cells that lose the plasmid at cell division. Such comparisons revealed a highly significant degree of similarity, suggesting a functional relationship. Based on these findings, it is proposed that A8 evolved from ahok-like protein, whose original role was the maintenance of an extrachromosomal replicon in the endosymbiont ancestor of mitochondria. An aggressive mechanism for the evolutionary maintenance of mitochondrial DNA overcomes many of the failings of traditional explanations for its retention as a separate genome.     

Identification of potent,selective, CNS-targeted inverse agonists of the ghrelin receptor

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine–piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.     

The pyoverdin receptor FpvA,a TonB-dependent receptor involved in iron uptake by Pseudomonas aeruginosa (Review)

Iron is an important element, essential for the growth of almost all living cells. Because of the high insolubility of iron(III) in aerobic conditions, many gram-negative bacteria produce, under iron limitation, small iron-chelating compounds called siderophores, together with new outer-membrane proteins, which function as receptors for the ferrisiderophores. Pseudomonas aeruginosa, an important human opportunistic pathogen, produces at least three known siderophores when grown in irondeficient conditions: pyochelin, salicylate and pyoverdin. This reviewfocuses on pyoverdin and on the ability of FpvA to bind iron-free and ferric-PaA pyoverdin, in the light of recent information gained from biochemical and biophysical studies and of the recently solved 3D-structures of the related ferrichrome FhuA and enterobactin FepA receptors in Escherichia coli.     

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45(low) c-Kit(+) cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45(low) c-Kit(-) cells that showed a granulocyte morphology; CD45(high) c-Kit(low/-) that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45(low) c-Kit(+) cells from the AGM culture had the abilities to reproduce CD45(low) c-Kit(+) cells and differentiate into CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) cells, whereas CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) did not produce CD45(low) c-Kit(+) cells. Furthermore, CD45(low) c-Kit(+) cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45(low) c-Kit(+) cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.     

Photopigment and receptor properties in Drosophila compound eye and ocellar receptors

A review of the spectral sensitivity and the rhodopsin and metarhodopsin characteristics in three compound eye receptor types (R1–6, R7, and R8) and ocellar receptors is presented (Fig. 1). Photopigment properties were determined from measures of conversion efficiency. The photopigments of R1–6 were studied using in vivo microspectrophotometry in the deep pseudopupil of white-eyed flies. These studies yielded a refined estimate of the R1–6 metarhodopsin spectrum (Fig. 2). The quantum efficiency relative to the spectral sensitivity estimate of the rhodopsin spectrum was factored out. The quantum efficiency of rhodopsin is about 1.75 times that of metarhodopsin. The peak absorbance of metarhodopsin was estimated to be about 2.6 times that of rhodopsin. The mechanism of the two-peaked R1–6 spectral sensitivity and metarhodopsin spectrum is discussed in terms of evidence that there is only one rhodopsin in R1–6 and that vitamin A deprivation preferentially lowers ultraviolet sensitivity. The prolonged depolarizing afterpotential is reviewed from the standpoint of the internal transmitter hypothesis of visual excitation. A careful comparison of the intensity-responsivity for photopigment conversion and its adaptional consequences is made (Fig. 3).     

Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.