Change of surface structure of poly(3-hydroxybutyrate) film upon enzymatic hydrolysis by PHB depolymerase

The change in the surface structure of poly[(R)-3-hydroxybutyrate] [PHB] films upon the enzymatic hydrolysis was analyzed by attenuated total reflection infrared [ATR/IR] spectrometry. As enzymes, PHB depolymerases isolated from Ralstonia pickettii T1 and Pseudomonas stutzeri were used. By curve decomposition of the carbonyl stretching band of ATR/IR spectra, the change in the surface crystallinity of PHB films by exposure to buffer containing 0, 1, and 4 microg of PHB depolymerases was estimated. It has been widely believed that the enzymatic hydrolysis first occurs in the amorphous phase, followed by the degradation in the crystalline phase, and extracellular PHB depolymerase can degrade only polymer chains in the surface layer of the film. Therefore, the surface crystallinity had been expected to increase upon the enzymatic degradation. However, the results were contrary to this expectation. The surface crystallinity was decreased by the enzymatic attack. Because ATR/IR spectrometry is sensitive to a small change in molecular structure of the sample surface, the decrease in the crystallinity shown by ATR/IR experiments probably does not indicate the complete loss of regularity of the crystalline phase. Because the chains at crystalline surface are more mobile than those inside the crystals, the C=O band for crystalline surface may appear at a position similar to those of the amorphous or interfacial phase in ATR/IR spectra of PHB. Only the chains inside the crystals may contribute to the C=O band of the crystalline phase. Thus, we rather suppose that the decrease in the crystalline peak of the ATR/IR spectra reflects the change in chain mobility or the increase of crystalline surface area by cracking of lamellas at the surface layers of PHB films or both.     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification.

Notch (N) and its ligands, Delta (Dl) and Serrate (Ser), are membrane-spanning proteins with EGF repeats. They play an essential role in mediating proliferation and segregated differentiation of stem cells. One of the prominent features of N signal system is that its ligands are anchored to the plasma membrane, which allows the ligand/receptor association only between the neighboring cells. Various lines of evidences have verified this intercellular signal transmission, but there also have been implications that expression of Dl or Ser interferes cell-autonomously with the ability of the cell to receive N signal, implying that N and its ligands may interact in the same cell. Here, we demonstrate that N, Dl, and Ser cell-autonomously form homomeric or heteromeric complexes. The cell-autonomous heteromeric complexes are not present on the cell surface, implying that the association occurs in the endoreticulum or Golgi apparatus. Expression of Dl or Ser cell-autonomously reduces the N-mediated HES-5 promoter activity, indicating that the cell-autonomous association alters the N signal receptivity. Intracellular deletion of Dl shows elevated activity of this dominant-negative effect. In vivo overexpression study suggests that the cell-autonomous function of Dl and Ser is independent of the ligand specificity and may be modulated by Fringe (Fg), which inhibits the formation of the cell-autonomous Dl/N or Ser/N complex.     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

A 63-base pair DNA segment containing an Sp1 site but not a canonical E2F site can confer growth-dependent and E2F-mediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7-related kinase

Cdc7-Dbf4 kinase complexes, conserved widely in eukaryotes, play essential roles in initiation and progression of the S phase. Cdc7 kinase activity fluctuates during cell cycle, and this is mainly the result of oscillation of expression of the Dbf4 subunit. Therefore, it is crucial to understand the mechanisms of regulation of Dbf4 expression. We have isolated and characterized the promoter region of the human ASK gene encoding Dbf4-related regulatory subunit for human Cdc7 kinase. We have identified a 63-base pair ASK promoter segment, which is sufficient for mediating growth stimulation. This minimal promoter segment (MP), containing an Sp1 site but no canonical E2F site, can be activated by ectopic E2F expression as well. Within the 63-base pair region, the Sp1 site as well as other elements are essential for stimulation by growth signals and by E2F, whereas an AT-rich sequence proximal to the coding region may serve as an element required for suppression in quiescence. Gel shift assays in the presence of an antibody demonstrate the presence of E2F1 in the protein-DNA complexes generated on the MP segment. However, the complex formation on MP was not competed by a DHFR promoter fragment, known to bind to E2F, nor by a consensus E2F binding oligonucleotide. Gel shift assays with point mutant MP fragments indicate that a non-canonical E2F site in the middle of this segment is critical for generation of the E2F complex. Our results suggest that E2F regulates the ASK promoter through an atypical mode of recognition of the target site.     

Fibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway

The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparin-like molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23. The present findings have revealed a novel MAPK-dependent mechanism of the regulation of phosphate uptake by FGF signaling.     

Colocalization and ligand-dependent discrete distribution of the estrogen receptor (ER)alpha and ERbeta

To investigate the relationships between the loci expressing functions of estrogen receptor (ER)alpha and that of ERbeta, we analyzed the subnuclear distribution of ERalpha and ERbeta in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP)-ERbeta redistributed from a diffuse to discrete pattern within the nucleus, showing a similar time course as FP-ERalpha, and colocalized with FP-ERalpha in the same discrete cluster. Analysis using deletion mutants of ERalpha suggested that the ligand-dependent redistribution of ERalpha might occur through a large part of the receptor including at least the latter part of activation function (AF)-1, the DNA binding domain, nuclear matrix binding domain, and AF-2/ligand binding domain. In addition, a single AF-1 region within ERalpha homodimer, or a single DNA binding domain as well as AF-1 region within the ERalpha/ERbeta heterodimer, could be sufficient for the cluster formation. More than half of the discrete clusters of FP-ERalpha and FP-ERbeta were colocalized with hyperacetylated histone H4 and a component of the chromatin remodeling complex, Brg-1, indicating that ERs clusters might be involved in structural changes of chromatin.     

Access to enantiomerically pure intermediates for (-)-geosmin synthesis starting from (4aS,5S)-4,4a,5,6,7,8-hexahydro-5-hydroxy-4a-methylnaphthalen-2(3H)-one

Enantiomerically pure key intermediates for the synthesis of the natural enantiomer of geosmin were synthesized from (4aS,5S)-4,4a,5,6,7,8-hexahydro-5-hydroxy-4a-methylnaphthalen-2(3H)-one.