Formation of spray-dried powder of S-adenosyl-L-methionine

S-Adenosyl-L -methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .     

Regulated spatial expression of fusion gene constructs with the 5′ upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5 flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for -galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another ascidian, namely Ciona savignyi. When a moderate amount of linearized pHrMA4a-Z was introduced into fertilized Ciona eggs, the expression of the reporter gene was evident in muscle cells of the larvae, suggesting that both species share a common machinery for the expression of muscle actin genes. The 5 upstream region of HrMA4a contains several consensus sequences, including a TATA box at -30, a CArG box at -116 and four E-boxes within a region of 200 bp. A deletion construct, in which only the 216-bp 5 flanking region of HrMA4a was fused with lac-Z, was expressed primarily in larval muscle cells. However, another deletion construct consisting of only the 61-bp upstream region of HrMA4a fused with lac-Z was not expressed at all. When pHrMA4a-Z or pHrMA4a-Z (–216) was injected into each of the muscle-precursor blastomeres of the 8-cell embryo, expression of the reporter gene was observed in larval muscle cells in a lineage-specific fashion. However, expression of the reporter gene was not observed when the plasmid was injected into non-muscle lineage. Therefore, the expression of the reporter gene may depend on some difference in cytoplasmic constituents between blastomeres of muscle and non-muscle lineage in the 8-cell embyo.     

The gene and deduced protein sequences of the zymogen of Aspergillus niger acid proteinase A

Proteinase A obtained from the culture medium of Aspergillus niger var. macrosporus is a unique acid endopeptidase that is insensitive (or less sensitive) to specific inhibitors of ordinary acid or aspartic proteinases, such as pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)-propane. In the preceding paper (Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S. B. P., Takahashi, T., Akanuma, H., Mamiya, G., Yamasaki, M. J. Biol. Chem. 266, 19480-19483), we reported the complete primary structure of the mature enzyme determined at the protein level. The enzyme has a unique two-chain structure with a 39-residue light (L) chain and a 173-residue heavy (H) chain linked noncovalently. As an extension of this study, we isolated genomic and cDNA clones encoding this proteinase and determined their nucleotide sequences. To isolate a genomic clone, the genomic DNA was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the amino acid sequence of the H chain, and a specific probe thus generated was used for screening a lambda gt10 genomic library. A cDNA for the enzyme was also selectively amplified by polymerase chain reaction using primers synthesized based on the sequence of the genomic DNA. Sequencing of the cloned genomic DNA and cDNA revealed the nucleotide sequence of the structural gene for the enzyme of 846 base pairs without introns. It encodes the precursor form of proteinase A, including an NH2-terminal preprosequence of 59 residues, the L chain of 39 residues, an intervening sequence of 11 residues, and the H chain of 173 residues linked in that order. Thus, proteinase A is thought to be synthesized as a single peptide chain preproenzyme of 282 residues, which is processed to generate the mature two-chain form.     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Development of the ovarian surface and associated germ cells in the human fetus

Summary The ovarian surface and associated germ cells have been studied in human fetuses from 12 weeks of age until near term, using light, TEM and SEM techniques. The surface epithelium and related cords proliferate extensively, especially at midterm. The cords in the ovarian cortex appear to be linked with ingrowths from the surface epithelium, and both structures have a common basal lamina. Germ cells are always interspersed among the somatic cells of the surface epithelium and associated cords. These results indicate that both the proliferating cords and surface epithelium may contribute to the formation of early follicles. Furthermore, the occurrence, of elements having some of the features of primitive steroidogenic cells in the regions of cordsurface epithelium continuity, suggests that both structures (surface epithelium and cords) contribute somatic cells, which in addition to becoming granulosa cells, might also contribute to the provision of primitive interstitial cells.Gonocytes tend to migrate through the developing ovarian tissue towards the surface where they become extruded into the peritoneal cavity. This phenomenon might contribute to the reduction in the number of germ cells at birth and parallels the atretic processes within the ovary.     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Native-State Heterogeneity of β2-Microglobulin as Revealed by Kinetic Folding and Real-Time NMR Experiments

The kinetic folding of β₂-microglobulin from the acid-denatured state was investigated by interrupted-unfolding and interrupted-refolding experiments using stopped-flow double-jump techniques. In the interrupted unfolding, we first unfolded the protein by a pH jump from pH 7.5 to pH 2.0, and the kinetic refolding assay was carried out by the reverse pH jump by monitoring tryptophan fluorescence. Similarly, in the interrupted refolding, we first refolded the protein by a pH jump from pH 2.0 to pH 7.5 and used a guanidine hydrochloride (GdnHCl) concentration jump as well as the reverse pH jump as unfolding assays. Based on these experiments, the folding is represented by a parallel-pathway model, in which the molecule with the correct Pro32 cis isomer refolds rapidly with a rate constant of 5–6 s^? 1, while the molecule with the Pro32 trans isomer refolds more slowly (pH 7.5 and 25 °C). At the last step of folding, the native-like trans conformer produced on the latter pathway isomerizes very slowly (0.001–0.002 s^? 1) into the native cis conformer. In the GdnHCl-induced unfolding assays in the interrupted refolding, the native-like trans conformer unfolded remarkably faster than the native cis conformer, and the direct GdnHCl-induced unfolding was also biphasic, indicating that the native-like trans conformer is populated at a significant level under the native condition. The one-dimensional NMR and the real-time NMR experiments of refolding further indicated that the population of the trans conformer increases up to 7–9% under a more physiological condition (pH 7.5 and 37 °C).     

The Molten Globule of β2-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

The acid transition of β₂-microglobulin (β2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH 4 (25 °C). The pH 4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20% larger than that of the native state. The accumulation of the pH 4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH 4 MG has been well characterized, although β2m, a β-protein, is structurally very different from α-helical apomyoglobin. NMR pH titration of histidine residues of β2m has also indicated that His84 has an abnormally low pK_a value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH 4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of β2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH 4 intermediate. It is proposed that the folding of β2m from its acid-denatured state takes place in the following order: denatured state → MG → burst-phase intermediate → native state.