Estrogen-mediated induction of a vitellogenin-specific nonhistone chromatin protein in the male chicken liver

Summary Non-histone chromatin proteins prepared from the livers of estrogen-treated and nontreated male chickens were compared by reverse-phase high performance liquid chromatography (RP-HPLC), followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that the hormone-treated male liver chromatin contained a specific protein corresponding to the vitellogenin-specific protein previously purified from the liver of egg-laying hens (Nakayama 1985). The chicken protein, purified further by gel-filtration high performance liquid chromatography (GF-HPLC), showed specific binding activity to DNA fragments carrying a part of the vitellogenin gene. On the basis of similarities in the elution patterns from GF-HPLC and RP-HPLC as well as in the mobility on SDS-PAGE, we concluded that this hormone-induced protein in the male chicken liver was identical to the vitellogenin-specific protein identified in the hen liver, and assumed it to be a specific regulatory protein for the vitellogenin gene expression. The amino acid composition of this chicken protein has been determined.     

An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

Characterization of the unfolding process of lipocalin-type prostaglandin D synthase

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.     

The epistatic interrelationships of IL-1, IL-1 receptor antagonist,and the type I IL-1 receptor

Mice lacking the gene for the IL-1R antagonist (IL-1ra) show abnormal development and homeostasis as well as altered responses to infectious and inflammatory stimuli. A reduction in the level of IL-1 signaling, either by deletion of the receptor or increased expression of IL-1ra, does not affect development or homeostasis, but does alter immune responses. In this study we use genetic epistasis to investigate the interdependence of selected genes in the IL-1 family in the regulation of these developmental and immunological processes. Deletion of the gene encoding the type I IL-1R (IL-1RI) is epistatic to deletion of the IL-1ra gene. Therefore, all functions of IL-1ra depend upon the presence of a functional receptor; there is no other target. Similarly, overexpression of the mRNA encoding the secreted form of IL-1ra is epistatic to deletion of the receptor antagonist, leaving the role of the intracellular splice variants of IL-1ra unknown. The abnormal development of IL-1ra-deficient mice is probably due to chronic overstimulation of the proinflammatory pathway via IL-1, but a clear single pathological defect is not apparent. These results support the model that the only essential function of IL-1ra in both health and disease is competitive inhibition of the IL-1RI.     

Fruit Development, Ripening and Quality Related Genes in the Papaya Genome

Papaya (Carica papaya L.) is the first fleshy fruit with a climacteric ripening pattern to be sequenced. As a member of the Rosids superorder in the order Brassicales, papaya apparently lacks the genome duplication that occurred twice in Arabidopsis. The predicted papaya genes that are homologous to those potentially involved in fruit growth, development, and ripening were investigated. Genes homologous to those involved in tomato fruit size and shape were found. Fewer predicted papaya expansin genes were found and no Expansin Like-B genes were predicted. Compared to Arabidopsis and tomato, fewer genes that may impact sugar accumulation in papaya, ethylene synthesis and response, respiration, chlorophyll degradation and carotenoid synthesis were predicted. Similar or fewer genes were found in papaya for the enzymes leading to volatile production than so far determined for tomato. The presence of fewer papaya genes in most fruit development and ripening categories suggests less subfunctionalization of gene action. The lack of whole genome duplication and reductions in most gene families and biosynthetic pathways make papaya a valuable and unique tool to study the evolution of fruit ripening and the complex regulatory networks active in fruit ripening.     

Molecular Cloning and Expression of a Novel Cholinephosphotransferase Involved in Glycoglycerophospholipid Biosynthesis of <Emphasis Type="Italic">Mycoplasma fermentans</Emphasis>

A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using α-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5′-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.     

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD₂)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys⁶⁵ residue and its network of hydrogen bonds with the polar residues Ser⁴⁵, Thr⁶⁷, and Ser⁸¹, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys⁶⁵ residue is activated by its interaction with Ser⁴⁵ and Thr⁶⁷ and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.Prostaglandin (PG)⁶ D synthase (PGDS; PGH₂ d-isomerase (EC 5.3.99.2)) (1, 2) produces PGD₂, having 9α-hydroxy and 11-keto groups, from PGH₂, which bears the chemically labile 9,11-endoperoxide group and is produced as a common intermediate of all prostanoids by the action of cyclooxygenase (PGH₂ synthase). Two distinct types of PGDS have evolved from phylogenetically distinct protein families (2, 3). One is hematopoietic PGDS (H-PGDS), which belongs to the σ class of GSH S-transferases (4, 5), and the other is lipocalin type PGDS (L-PGDS), a member of the lipocalin family (6, 7). L-PGDS is the only enzyme in the lipocalin family and is identical to β-trace, a major protein in human cerebrospinal fluid (8, 9). Although H-PGDS and L-PGDS catalyze the same reaction, their amino acid sequences and tertiary structures are quite different from each other, indicating that these enzymes are a new example of functional convergence (2, 3).L-PGDS is expressed in the heart, central nervous system, and male genital organs of various mammals and is involved in various physiological and pathological functions (reviewed in Refs. 6 and 7). In the brain, L-PGDS produces PGD₂, which is involved in the regulation of pain and non-rapid eye movement sleep, as was shown in studies using gene knock-out mice (10, 11) and human enzyme transgenic mice (12). L-PGDS is regulated by SOX9 and is involved in the differentiation of male genital organs (13–15). This enzyme is also expressed in adipocytes (16), vascular smooth muscle cells (17), and myocardial cells (18, 19) and is involved in adipocyte differentiation, the progression of arteriosclerosis (20), and the protection against hypoxemia (18) or ischemia/reperfusion injury (19). L-PGDS binds various lipophilic compounds, such as retinoids (21), bilirubin, biliverdin (22), gangliosides (23), and amyloid-β peptides (24, 25), with high affinity, acting as an extracellular transporter of these compounds and serving as an endogenous amyloid-β chaperone to prevent amyloid deposition in vivo (24).Although many biochemical and physiological studies suggest important roles of PGD₂ and L-PGDS/β-trace in the regulation of sleep and other biological functions, the crystal structure of L-PGDS has not been resolved. In this study, we determined the crystal structures of two different forms of the Δ^1–24-C65A mutant of mouse L-PGDS in both open and closed conformations. L-PGDS was shown to possess a typical lipocalin fold, the β-barrel, which is a unique structural component specific to L-PGDS and comprises a mobile E-F loop and a large central cavity with two compartments. By performing site-directed mutagenesis of Δ^1–24-L-PGDS and the Δ^1–24-C65A mutant, we found that the Cys⁶⁵ surrounded by the hydroxyl side chains of Ser⁴⁵, Thr⁶⁷, and Ser⁸¹ was activated to contribute to the catalysis by L-PGDS.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.