Purification and characterization of acid phosphatase-1 from Drosophila melanogaster

Acid phosphatase-1 (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2), the major phosphatase in adult Drosophila melanogaster, has been purified to apparent homogeneity. The final product is a glycoprotein homodimer with a subunit molecular weight of about 50,000, as measured by its electrophoretic mobility in denaturing conditions on polyacrylamide gels containing sodium dodecyl sulfate. It has a turnover number of 1720 1-naphthyl phosphate molecules hydrolyzed/s by each acid phosphatase-1 molecule at 37 degrees C, pH 5.0. An average fly contains about 5 ng of enzyme. Pure acid phosphatase-1 displays heterogeneity in isoelectric focusing, with a major band at pH 5.3. The enzyme hydrolyzes a wide variety of phosphate monoesters, including AMP, glucose 6-phosphate, ATP, choline phosphate, or phosphoproteins. The maximum reaction rates are different for all substrates, and some substrates appear to inhibit the reaction at high substrate concentrations. The Michaelis constants for 1-naphthyl phosphate and p-nitrophenyl phosphate are 79 microM and 68 microM, respectively, at pH 5.0 and 37 degrees C. The optimum pH level for 1-naphthyl phosphate is 4.5. Acid phosphatase-1 is inhibited by L(+)-tartrate (but not D(-)-tartrate), phosphate, and fluoride. The reaction rate increases 2.1-fold for every 10 degrees C rise in temperature. Above 48 degrees C, the rate of thermal denaturation is greater than the rate of the enzyme reaction.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Functional characterization and genetic mapping of alk8

The novel type I TGFβ family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.     

Evolutionary developmental biology and genomics

Reciprocal questions often frame studies of the evolution of developmental mechanisms. How can species share similar developmental genetic toolkits but still generate diverse life forms? Conversely, how can similar forms develop from different toolkits? Genomics bridges the gap between evolutionary and developmental biology, and can help answer these evo-devo questions in several ways. First, it informs us about historical relationships, thus orienting the direction of evolutionary diversification. Second, genomics lists all toolkit components, thereby revealing contraction and expansion of the genome and suggesting mechanisms for evolution of both developmental functions and genome architecture. Finally, comparative genomics helps us to identify conserved non-coding elements and their relationship to genome architecture and development.     

Desferrioxamine inhibits protein tyrosine nitration: Mechanisms and implications

Tissues are exposed to exogenous and endogenous nitrogen dioxide (()NO(2)), which is the terminal agent in protein tyrosine nitration. Besides iron chelation, the hydroxamic acid (HA) desferrioxamine (DFO) shows multiple functionalities including nitration inhibition. To investigate mechanisms whereby DFO affects 3-nitrotyrosine (3-NT) formation, we utilized gas-phase ()NO(2) exposures, to limit introduction of other reactive species, and a lung surface model wherein red cell membranes (RCM) were immobilized under a defined aqueous film. When RCM were exposed to ()NO(2) covered by +/- DFO: (i) DFO inhibited 3-NT formation more effectively than other HA and non-HA chelators; (ii) 3-NT inhibition occurred at very low[DFO] for prolonged times; and (iii) 3-NT formation was iron independent but inhibition required DFO present. DFO poorly reacted with ()NO(2) compared to ascorbate, assessed via ()NO(2) reactive absorption and aqueous-phase oxidation rates, yet limited 3-NT formation at far lower concentrations. DFO also inhibited nitration under aqueous bulk-phase conditions, and inhibited 3-NT generated by active myeloperoxidase "bound" to RCM. Per the above and kinetic analyses suggesting preferential DFO versus ()NO(2) reaction within membranes, we conclude that DFO inhibits 3-NT formation predominantly by facile repair of the tyrosyl radical intermediate, which prevents ()NO(2) addition, and thus nitration, and potentially influences biochemical functionalities.     

Molecular pedomorphism underlies craniofacial skeletal evolution in Antarctic notothenioid fishes

Background  

Pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. Despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. Notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing Antarctic climate. Notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy.     

Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary

Background  

The processes by which eggs develop in the insect ovary are well characterized. Despite a large number of Drosophila mutants that cannot lay eggs, the way that the egg is moved along the reproductive tract from ovary to uterus is less well understood. We remedy this with an integrative study on the reproductive tract muscles (anatomy, innervation, contractions, aminergic modulation) in female flies.     

Characterization of the retinoic acid receptor genes raraa, rarab and rarg during zebrafish development

Retinoic acid signaling is important for patterning the central nervous system, paired appendages, digestive tract, and other organs. To begin to investigate retinoic acid signaling in zebrafish, we determined orthologies between zebrafish and tetrapod retinoic acid receptors (Rars) and examined the expression patterns of rar genes during embryonic development. Analysis of phylogenies and conserved syntenies showed that the three cloned zebrafish rar genes include raraa and rarab, which are co-orthologs of tetrapod Rara, and rarg, which is the zebrafish ortholog of tetrapod Rarg. We did not, however, find an ortholog of Rarb. RNA in situ hybridization experiments showed that rarab and rarg, are maternally expressed. Zygotic expression of raraa occurs predominantly in the hindbrain, lateral mesoderm, and tailbud. Zygotic expression of rarab largely overlaps that of raraa, except that in later stages rarab is expressed more broadly in the brain and in the pectoral fin bud and pharyngeal arches. Zygotic expression of zebrafish rarg also overlaps the other two genes, but it is expressed more strongly in the posterior hindbrain beginning in late somitogenesis as well as in neural crest cells in the pharyngeal arches. Thus, these three genes have largely overlapping expression patterns and a few gene-specific expression domains. Knowledge of these expression patterns will guide the interpretation of the roles these genes play in development.     

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.