Suppression of female-specific murine Cyp2b9 gene expression by growth or glucocorticoid hormones

CYP2B9 is a constitutively and female-specifically expressed P450 isoform in mouse livers. Hypophysectomy-induced CYP2B9 mRNA expression in males to a level similar to that in females, while the operation did not affect females. Twice-daily injection of growth hormone (GH), which mimics the male pattern of GH secretion, significantly repressed hypophysectomy-induced mRNA expression in males. The same treatment completely suppressed expression in intact females. Treatments with synthetic glucocorticoid dexamethasone (DEX) suppressed expression of CYP2B9 mRNA in intact females, but not in GH-treated and un-treated hypophysectomized females. In primary cultured mouse hepatocytes, CYP2B9 mRNA expression was concentration-dependently suppressed by natural glucocorticoids such as hydrocortisone and corticosterone as well as by DEX. Glucocorticoid-mediated suppression was partially inhibited by RU486, a potent antiglucocorticoid. In contrast, RU486 by itself suppressed expression of CYP2B9 mRNA. These observations suggest that the sexually dimorphic expression of CYP2B9 is partly due to suppression by the masculine plasma GH profile and by glucocorticoid hormones.     

Phenylethanoid glycosides from Phlomis integrifolia Hub.-Mor

Two new phenylethanoid glycosides integrifoliosides A (2) and B (3), along with a known phenylethanoid glycoside alyssonoside (1) and a flavone glucoside chrysoeriol-7-O-beta-D-glucopyranoside (4) were isolated from the aerial parts of Phlomis integrifolia. The structures of the new compounds were identified as 3,4-dihydroxy-beta-phenylethoxy-O-beta-D-apiofuranosyl-(1 --> 4)-alpha-L-rhamnopyranosyl-(1 --> 3)-4-O-feruloyl-beta-D-glucopyranoside (2) and 3-hydroxy-4-methoxy-beta-phenylethoxy-O-beta-D-apiofuranosyl-(1 --> 4)-alpha-L-rhamnopyranosyl-(1 --> 3)-4-O-feruloyl-beta-D-glucopyranoside (3), on the basis of spectroscopic (UV, IR, 1D- and 2D-NMR, and HR-FABMS) methods.     

Synthetic studies on glycosphingolipids from the Protostomia phyla: syntheses of arthro-series glycosphingolipids

Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcbeta-(1 --> 3)-Man and GalNAcbeta-(1 --> 4)-GlcNAcbeta-(1 --> 3)-Man. We have synthesized two glycosphingolipids, beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer and beta-D-GalNAcp-(1 --> 4)-beta-D-GlcNAcp-(1 --> 3)-beta-D-Manp-(1 --> 4)-beta-D-Glcp-(1 --> 1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for the synthesis of the beta-mannopyranosidic linkages.     

In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation

Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, alpha-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated alpha-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules.     

Fibronectin peptides derived from two distinct regions stimulate adipocyte differentiation by preventing fibronectin matrix assembly

Here, we show that fibronectin (FN) peptides derived from two distinct regions promote the insulin-induced adipocyte differentiation of ST-13 cells by preventing FN fibrillogenesis. ST-13 cells formed numerous FN fibrils under nonadipogenic conditions, whereas this FN fibrillogenesis was suppressed by adipose induction with insulin. The insulin-induced adipocyte differentiation was promoted by an amino-terminal 24-kDa fragment of FN, accompanied by further suppression of FN fibrillogenesis. The 24 K fragment prevented FN matrix assembly by direct incorporation into the FN matrix. Like the 24 K fragment, a peptide from the 14th type III repeat, termed FNIII14, which suppressed the integrin alpha 5 beta 1-mediated adhesion of ST-13 cells to FN, accelerated the adipocyte differentiation by preventing FN fibrillogenesis without direct incorporation into the FN matrix. FNIII14 induced the conformation change of beta1 integrins of K562 cells from active to resting, as judged by FACS analysis using a monoclonal antibody AG89 directed to an active beta1 integrin-dependent epitope. Binding of a (125)I-labeled FN fragment containing the RGD cell adhesive site to ST-13 cell surface was dissociated by FNIII14, with a concomitant binding of FNIII14 itself to the cell surface. The affinity labeling of ST-13 cells using biotinylated FNIII14 showed that FNIII14 specifically bound to a nonintegrin membrane protein with M(r) of around 50 kDa. Thus, the results indicated that prevention of FN fibrillogenesis by the 24 K Fib 1 fragment and FNIII14 caused the promotion of adipocyte differentiation of ST-13 cells and that the former was due to the direct incorporation into the FN matrix and that the latter might be interpreted by negative regulation of FN receptor alpha 5 beta 1 activity.     

Chromosomal variations within aneuploid cancer lines.

Aneuploid cancers exhibit a wide spectrum of clinical aggressiveness, possibly because of varying chromosome compositions. To test this, karyotypes from the diploid CCD-34Lu fibroblast and the aneuploid A549 and SUIT-2 cancer lines underwent fluorescence in situ hybridization (FISH) and DAPI counterstaining. The number of DAPI-stained and FISH-identified chromosomes, 1-22, X,Y, as well as structural abnormalities, were counted and compared using the chi(2), Mann-Whitney rank sum test and the Levene's equality of variance. Virtually all of the evaluable diploid CCD-34Lu karyotypes had 46 chromosomes with two normal-appearing homologues. The aneuploid chromosome numbers per karyotype were highly variable, averaging 62 and 72 for the A549 and SUIT-2 lines, respectively. However, the A549 chromosome numbers were more narrowly distributed than the SUIT-2 karyotype chromosome numbers. Furthermore, 25% of the A549 chromosomes had structural abnormalities compared to only 7% of the SUIT-2 chromosomes. The chromosomal compositions of the aneuploid A549 and SUIT-2 cancer lines are widely divergent, suggesting that diverse genetic alterations, rather than chance, may govern the chromosome makeups of aneuploid cancers.     

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways

To investigate the mechanism of theregulation of human red blood cell deformability, we examined thedeformability under mechanical stress. Washed human red blood cellswere rapidly injected through a fine needle, and their filterabilitywas measured using a nickel mesh filter. The decrease in filterabilityshowed a V-shaped curve depending on the extracellularCa²⁺ concentration; the maximumdecrease was achieved at ~50 µM. The decreased filterability wasaccompanied by no change in cell morphology and cell volume, indicatingthat the decrease in filterability can be ascribed to alterations ofthe membrane properties. Ca²⁺entry blockers (nifedipine and felodipine) inhibited the impairment offilterability under mechanical stress. ProstaglandinsE₁ and E₂, epinephrine, andpentoxifylline, which are thought to modulate the intracellularadenosine 3',5'-cyclic monophosphate (cAMP) level of redblood cells, improved or worsened the impaired filterability accordingto their expected actions on the cAMP level of the cells. These resultsstrongly suggest that the membrane properties regulating red blood celldeformability are affected by the signal transduction system, includingCa²⁺-dependent and cAMP-mediatedsignaling pathways.

     

Enhancement of Human Immunodeficiency Virus Type 1 (HIV-1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV-1 infection. Although virus-cell binding was significantly inhibited by PEI, HIV-1 infection in human T-cell lines such as MT-4 and MOLT-4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV-1 infection was examined using HIV-1 chronically infected cells (MOLT-4/HIV-1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT-4/HIV-1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV-1 to CD4-positive cells by PEI was due to the shedding of gp120 from HIV-1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV-1 infection. Similarly, dissociation of gp120 from MOLT-4/HIV-1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV-1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.     

Mechanism of inhibition of Chromatium D growth by L-methionine regulation of L-threonine biosynthesis by the intracellular level of S-adenosylmethionine

1. (1) An unusual accumulation of S-adenosyl-L-methionine in Chromatium D was associated with a marked growth inhibition by L-methionine. The inhibition was overcome by L-isoleucine, L-leucine, L-phenylalanine, L-threonine, L-valine and putrescine. Based on their effects, these compounds are classified into 3 types.

2. (2) L-Isoleucine, L-leucine, L-phenylalanine and L-valine (Type I) inhibited the L-methionine uptake and consequently prevented the bacterium from the unusual accumulation of S-adenosyl-L-methionine even in the presence of L-methionine in the medium. Putrescine (Type II) stimulated the consumption of S-adenosyl-L-methionine, but did not influence the L-methionine uptake. Hence, the effect of putrescine would be explained by the action to diminish the intracellular level of S-adenosyl-L-methionine. L-Threonine (Type III) neither inhibited the L-methionine uptake nor affected the content of S-adenosyl-L-methionine due to the addition of L-methionine.

3. (3) The specific activity of homoserine kinase (EC 2.7.1.39) was greatly lowered by the addition of L-methionine under conditions in which Chromatium D unusually accumulates S-adenosyl-L-methionine. Homoserine dehydrogenase (EC 1.1.1.3) activity was inhibited by S-adenosyl-L-methionine (50% inhibition index, 3.5 mM). These facts strongly suggest that the growth inhibition by L-methionine is associated with the L-threonine deficiency caused by the unusual accumulation of S-adenosyl-L-methionine.

Asparagusic acid, a new plant growth inhibitor in etiolated young asparagus shoots

Yellow prisms of asparagusic acid, with a molecular formulaof C₄H₆O₂S₂ were isolated from etiolated asparagus tissues (Asparagusofficinalis L.). This acid inhibits growth in lettuce and otherseedlings when applied in concentrations of 6.67x10^–7Mto 6.67xl0^–7M. The extent of activity was very similarto that of abscisic acid. ¹ A well known shift reagent in the NMR spectrum (1). (Received April 12, 1972; )