The evolution of sex-independent transmission ratio distortion involving multiple allelic interactions at a single locus in rice

Transmission ratio distortion (TRD) is frequently observed in inter- and intraspecific hybrids of plants, leading to a violation of Mendelian inheritance. Sex-independent TRD (siTRD) was detected in a hybrid between Asian cultivated rice and its wild ancestor. Here we examined how siTRD caused by an allelic interaction at a specific locus arose in Asian rice species. The siTRD is controlled by the S(6) locus via a mechanism in which the S(6) allele acts as a gamete eliminator, and both the male and female gametes possessing the opposite allele (S(6)(a)) are aborted only in heterozygotes (S(6)/S(6)(a)). Fine mapping revealed that the S(6) locus is located near the centromere of chromosome 6. Testcross experiments using near-isogenic lines (NILs) carrying either the S(6) or S(6)(a) alleles revealed that Asian rice strains frequently harbor an additional allele (S(6)(n)) the presence of which, in heterozygotic states (S(6)/S(6)(n) and S(6)(a)/S(6)(n)), does not result in siTRD. A prominent reduction in the nucleotide diversity of S(6) or S(6)(a) carriers relative to that of S(6)(n) carriers was detected in the chromosomal region. These results suggest that the two incompatible alleles (S(6) and S(6)(a)) arose independently from S(6)(n) and established genetically discontinuous relationships between limited constituents of the Asian rice population.     

小麦背景中簇毛麦6V染色体的遗传稳定性及其在配子中的传递

小麦－簇毛麦６Ｖ二体附加系,６Ａ（６Ｖ）二体代换系,６Ａ＾Ｌ．６Ｖ＾ｓ二体易位系在细胞学上是基本稳定的６Ｖ,６Ａ＾Ｌ．６Ｖ＾ｓ染色体能够通过配子稳定地传递给后代,在杂合状态下,带有６Ｖ的配子传递率普遍显著下降,在单体附加系（２１”Ｗ＋６Ｖ）中,６Ｖ通过雌配子的传递率（１１．３％）高于通过雄配子的传递率（５．９％）,在单体代换系（２０”Ｗ＋６Ａ－６Ｖ）中,６Ｖ通过雌配子的传递率（１０．４％）低于通过     

The effect of interleukin-6 (IL-6)/gp130 signalling on biliary epithelial cell growth, in vitro

The effect of IL-6 on the growth of mouse biliary epithelial cells (BEC), in vitro, was tested by comparing BEC obtained IL-6-deficient mice (IL-6(-/-)) to wild-type littermate controls (IL-6(+/+)), in two different media: simple serum-free media (S-SFM), and complete serum-free media (C-SFM) containing forskolin, which stimulates BEC IL-6 production. In S-SFM, neither IL-6(+/+)nor IL-6(-/-)BEC constitutively produced IL-6 mRNA or protein, and there was no difference between IL-6(+/+)and IL-6(-/-)BEC growth. In contrast, when the BEC were maintained in C-SFM, over 48 h, the growth of IL-6(+/+)BEC was 40% greater than IL-6(-/-)BEC (P<0.006). Enhanced IL-6(+/+)BEC growth in C-SFM was associated with induced expression of IL-6 mRNA and IL-6 protein secretion into the medium, upregulation of the IL-6Ralpha (gp80) and phosphorylation of the signal transducing molecule gp130. In C-SFM, anti-IL-6 neutralizing antibodies blocked enhanced IL-6(+/+)BEC growth, whereas exogenous rhIL-6 stimulated retarded growth of IL-6(-/-)BEC. Thus, under conditions that mimic an inflammatory or stressful microenvironment in vivo, BEC produce, secrete and respond to IL-6, via upregulation and activation of the IL-6Ralpha (gp80)/gp130 signaling system in an autocrine/paracrine manner.     

Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K(m) (20-23 μM) and k(cat) (4200-5100 s(-1)) values. Likewise, the K(i) values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone- and rod-specific PDE6 γ-subunits (Pγ) were comparable. Recombinant cone transducin-α (Gα(t2)) and native rod Gα(t1) fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of Gα(t2) or Gα(t1). Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with Pγ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.     

Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase.

Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.     

Conformational analysis of a ribopentanucleoside tetraphosphate in aqueous solution. A two-dimensional NMR study at 500 MHz.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions. The coupling constants of the ribose-ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta and gamma are presented. The combined data suggest a strong similarity between the 5'-terminal triplets in m6(2)AUm6(2)AUm6(2)A, m6(2)AUm6(2)AU and m6(2)AUm6(2)A2.     

Mutagenic and carcinogenic actions of some polycyclic aromatic hydrocarbons]

The carcinogenic and mutagenic actions of benz(a)pyrene (BP) and its derivatives 6-methyl-, 6-formyl-, 6-chloro-, 6-hydroxy-, 6-acetoxy-, 6-methoxy- and 4(5)-methoxy-BP were studied in mice and bacteria Salmonella typhimurium TA-98 and TA-100, respectively. The potent carcinogenic agents BP, 6-methyl-, 6-formyl-BP proved also mutagenic in bacteria of both strains. Weak carcinogenic compounds (6-chloro-, 6-methoxy-, 4(5)-methoxy-BP) and noncarcinogenic ones (6-acetoxy-, 6-hydroxy-BP) either turned out nonmutagenic or mutagenic in one of two bacterial strains tested. The differences in carcinogenic and mutagenic actions of the substances under study are not relative to the rate of their oxidation in the enzymatic system.     

Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulus P6 Biphenyl Dioxygenase and of Chimeras Derived from It

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The alpha or beta subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), ht-alpha(LB400)beta(P6), and ht-alpha(B-356)beta(P6). ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356) were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase alpha subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E. coli. Furthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an alpha and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.     

Role of growth hormone in modulating the constitutive and phenobarbital-induced levels of two P-450(6)beta (testosterone 6 beta-hydroxylase) mRNAs in rat livers

The role of growth hormone in the expression of two forms of hepatic cytochrome P-450(P-450), P-450(6)beta-1(6 beta-3), and P-450(6)beta-4, was investigated using RNA blots. The level of P-450(6)beta-1(6 beta-3) mRNA was twenty times higher than that of P-450(6) beta-4 mRNAs in untreated male rat livers. The levels of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were increased two fold and three fold, respectively, by hypophysectomy of adult male rats. By intermittent injection of human growth hormone (hGH) into hypophysectomized male rats, both mRNAs were decreased to the level of normal rats, and almost disappeared after continuous infusion of hGH. In female rats, these two mRNAs were not detected, but were increased remarkably by hypophysectomy. The increases in these mRNAs were almost abolished after continuous infusion of hGH in hypophysectomized female rats. The effect of hGH on PB-mediated induction of P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs was also examined. The PB-mediated increases in P-450(6)beta-1(6 beta-3) and P-450(6)beta-4 mRNAs were higher in hypophysectomized male rats (2.5-fold and 10.9-fold, respectively) than in normal male rats (1.5-fold and 5.2-fold, respectively). Thus, the levels of P-450(6)beta-1(6-beta-3) and P-450(6)beta-4 mRNAs were 4.1-fold and 7.3-fold, respectively, higher in PB-induced hypophysectomized rats than in normal male rats. Concerning the postnatal developmental profiles, P-450(6)beta-1(6 beta-3) mRNA was detectable at neonate and reached a maximal level at around 17 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transferring the methyl group from the 6-position of guanine to a cysteine residue on the protein. We previously found that the Escherichia coli Ada protein makes critical interactions with O6-methylguanine (O6mG) at the N1- and O6-positions. Human AGT has a different specificity than the bacterial protein. We reacted hAGT with double-stranded pentadecadeoxynucleotides containing analogues of O6mG. The second-order rate constants were in the following order (x10(-)5 M-1 s-1): O6mG (1.4), O6-methylhypoxanthine (1.6) > Se6-methyl-6-selenoguanine (0.1) > S6-methyl-6-thioguanine (S6mG) (0.02) > S6-methyl-6-thiohypoxanthine (S6mH), O6-methyl-1-deazaguanine (O6m1DG), O6-methyl-3-deazaguanine (O6m3DG), and O6-methyl-7-deazaguanine (O6m7DG) (all <0.0001). Electrophoretic mobility shift assays were carried out to determine the binding affinity to hAGT. Oligodeoxynucleotides containing O6mG, S6mG and O6m3DG bound to AGT in the presence of competitor DNA with Kd values from 5 to 20 microM, while those containing G, S6mH, O6m1DG, and O6m7DG did not (Kd > 200 microM). These results indicate that the 1-, N2-, and 7- positions of O6mG are critical in binding to hAGT, while the 3- and O6-positions are involved in methyl transfer. These results suggest that the active site of ada AGT is more flexible than hAGT and may be the reason ada AGT reacts with O4mT faster than hAGT.     

Conformational preferences of modified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) adenine and 2-methylthio-N6-(N-threonylcarbonyl) adenine by the quantum chemical PCILO calculations

Conformational preferences of the hypermodified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) Adenine, m6tc6 Ade, and 2-methylthio-N6-(N-threonylcarbonyl) Adenine, mS2 tc6 Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m6 tc6 Ade and mS 2tc6 Ade alike the threonylcarbonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N6-(N-threonylcarbonyl) Adenine, tc6 Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc6 Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(1) of the adenine base. The N6-methyl protons, in m6 tc6 Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS 2tc6 Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N6-(delta 2-isopentenyl) Adenine, mS 2i6 Ade, where the 2-methylthio group orients trans to the C(2)-N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

Bipyrimidine ruthenium(II) arene complexes: structure, reactivity and cytotoxicity

The synthesis and characterization of complexes [(η(6)-arene)Ru(N,N')X][PF(6)], where arene is para-cymene (p-cym), biphenyl (bip), ethyl benzoate (etb), hexamethylbenzene (hmb), indane (ind) or 1,2,3,4-tetrahydronaphthalene (thn), N,N' is 2,2'-bipyrimidine (bpm) and X is Cl, Br or I, are reported, including the X-ray crystal structures of [(η(6)-p-cym)Ru(bpm)I][PF(6)], [(η(6)-bip)Ru(bpm)Cl][PF(6)], [(η(6)-bip)Ru(bpm)I][PF(6)] and [(η(6)-etb)Ru(bpm)Cl][PF(6)]. Complexes in which N,N' is 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione or 4,7-diphenyl-1,10-phenanthroline (bathophen) were studied for comparison. The Ru(II) arene complexes undergo ligand-exchange reactions in aqueous solution at 310?K; their half-lives for hydrolysis range from 14 to 715?min. Density functional theory calculations on [(η(6)-p-cym)Ru(bpm)Cl][PF(6)], [(η(6)-p-cym)Ru(bpm)Br][PF(6)], [(η(6)-p-cym)Ru(bpm)I][PF(6)], [(η(6)-bip)Ru(bpm)Cl][PF(6)], [(η(6)-bip)Ru(bpm)Br][PF(6)] and [(η(6)-bip)Ru(bpm)I][PF(6)] suggest that aquation occurs via an associative pathway and that the reaction is thermodynamically favourable when the leaving ligand is I?>?Br?≈?Cl. pK (a)* values for the aqua adducts of the complexes range from 6.9 to 7.32. A binding preference for 9-ethylguanine (9-EtG) compared with 9-ethyladenine (9-EtA) was observed for [(η(6)-p-cym)Ru(bpm)Cl][PF(6)], [(η(6)-hmb)Ru(bpm)Cl](+), [(η(6)-ind)Ru(bpm)Cl](+), [(η(6)-thn)Ru(bpm)Cl](+), [(η(6)-p-cym)Ru(phen)Cl](+) and [(η(6)-p-cym)Ru(bathophen)Cl](+) in aqueous solution at 310?K. The X-ray crystal structure of the guanine complex [(η(6)-p-cym)Ru(bpm)(9-EtG-N7)][PF(6)](2) shows multiple hydrogen bonding. Density functional theory calculations show that the 9-EtG adducts of all complexes are thermodynamically preferred compared with those of 9-EtA. However, the bmp complexes are inactive towards A2780 human ovarian cancer cells. Calf thymus DNA interactions for [(η(6)-p-cym)Ru(bpm)Cl][PF(6)] and [(η(6)-p-cym)Ru(phen)Cl][PF(6)] consist of weak coordinative, intercalative and monofunctional coordination. Binding to biomolecules such as glutathione may play a role in deactivating the bpm complexes.     

Thyroid hormones and the reactivities of genetic variants of human erythrocytic glucose-6-phosphate dehydrogenase

Thyroid hormones, throxine (T4) and triiodothyronine (T3) which are known to activate glucose-6-phosphate dehydrogenase (G6PD) activity in vivo act as substrate inhibitors of G6PD in vitro. T4 competitively inhibits NADP in human erythrocyte G6PD variants G6PDA, G6PDB and G6PDA- with inhibition constants of 2.40 +/- 0.90 X 10(-6), 3.44 +/- 0.63 X 10(-6) and 6.53 +/- 0.60 X 10(-6) mol/l, respectively. The inhibition is, however, noncompetitive with respect to G6P in the three variants. T3 also has similar inhibition pattern to T4 with inhibition constants for NADP of 1.9 +/- 0.08 X 10(-5) and 1.28 +/- 0.17 X 10(-5) mol/l for G6PDB and G6PDA-, respectively. cAMP on the other hand inhibits G6P competitively with inhibition constants 1.50 +/- 0.22 X 10(-4), 1.06 +/- 0.24 X 10(-4) and 1.76 +/- 0.14 X 10(-4) mol/l for G6PDB, G6PDA and G6PDA-, respectively. There are significant differences in the inhibition effects of T4 and cAMP with respect to NADP as substrates for the normal enzyme G6PDA or G6PDB and the deficient enzyme G6PDA- when NADP is the substrate, the latter being much more inhibited. The activation effect of thyroid hormones in vivo may therefore not be a direct result of thyroid hormone binding to the G6PD enzyme nor mediated through the action of cAMP but plausibly be through complexation of inhibitory trace metal ions by the thyroid hormones T4 and T3.     

Glucose-6-Phosphate Dehydrogenase/6-Phosphogluconate Dehydrogenase Ratio and the Glucose-6-Phosphate, 6-Phosphogluconate and Fructose-6-Phosphate Contents in Tobacco Plants Infected with Potato Virus Y

The ratio of activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G6P DH/6PG DH), and the contents of glucose-6-phosphate (G6P), 6-phosphogluconate (6PG) and fructose-6-phosphate (F6P) were studied at various stages of potato virus Y (PVY) multiplication in Nicotiana tabacum cv. Samsun. G6P DH/6PG DH increased through the experiment from 0.42 to 0.53 in leaves of healthy tobacco, and up to 0.59 in PVY systemically infected leaves. However, these ratios in the ruptured protoplast preparations, and the chloroplast and cytosol fractions of healthy protoplasts were similar to that from infected ones. The ratio lower than 1, found in the healthy and/or PVY- infected leaf tissues and in the infected protoplasts as well, confirms the assumption that G6P DH is the control enzyme of oxidative pentosephosphate pathway not only in the healthy but also in the infected plants. The contents of G6P, 6PG and F6P in the period of the highest PVY multiplication were strongly decreased (to 30 – 50 % when compared with control healthy leaves) and were negatively correlated with the G6P DH and 6PG DH activities.     

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.     

Anti-peptide antibodies for examining the conformation, molecular assembly and localization of an intracellular protein, ribosomal protein S6, in vivo

Ribosomal protein S6 (rpS6) is known to relate to cell proliferation. Our recent proteome analysis of human metaphase chromosomes revealed the enrichment of rpS6 during mitosis. Here, structure, localization and molecular assembly in vitro and in vivo of a human rpS6, were examined using antibodies (Abs) prepared by immunizing rabbits with synthetic peptides. Five peptides, Ser6-Asp20 (S6-1), Ile52-Gly66 (S6-2), Asp103-Gly117 (S6-3), Asn146-Lys160 (S6-4) and Arg178-Ile192 (S6-5) were chosen as epitopes of human rpS6. These peptides except for S6-3 induced strong Ab production, and with an enzyme-linked immunosorbent assay, anti-S6-2, anti-S6-4 and anti-S6-5, showed high reactivity to recombinant rpS6 (r-rpS6), while anti-S6-1 did not, suggesting that S6-2, S6-4 and S6-5 were exposed on the r-rpS6 surface, while S6-1 was less exposed or possessed a different conformation. The immunostaining of HeLa cells as well as isolated chromosomes suggested that rpS6 occurs in endoplasmic reticulum (ER) but less possible on chromosomes since no Abs showed localization of rpS6 to chromosomes. In addition, the immunostaining suggested that only S6-4 is exposed on the protein surface, while S6-2 and S6-5 are buried by the interaction with other macromolecules in HeLa cells. Present our result shows new possibility of antibodies as tools for structure-oriented cell biology.     

Absorption and excretion of orally administered inositol hexaphosphate (IP(6) or phytate) in humans.

A study of the pharmacokinetic profile (oral absorption and renal excretion) of inositol hexaphosphate or phytate (IP(6)) is presented. Seven healthy volunteers were following a IP(6) poor diet (IP(6)PD) in a first period, and on IP(6) normal diet (IP(6)ND) in a second one. When following the IP(6)PD they become deficient in IP(6), the basal levels found in plasma (0.07+/- 0.01 mg/L) being clearly lower than those found when IP(6)ND was consumed (0.26+/- 0.03 mg/L). During the restriction period the maximum concentration in plasma were obtained 4 h after the ingestion of a single dose of IP(6), observing almost the same renal excretion profiles for the three different commercial sources and doses. After the IP(6) restriction period, volunteers were on IP(6)ND, reaching normal plasma and urinary IP(6) values in 16 days. Thus, the normal plasma and urinary concentrations, can be obtained either by consumption of a IP(6)ND taking a long time or in a short period by IP(6) supplements.     

Soluble human interleukin-6-receptor modulates interleukin-6-dependent N-glycosylation of alpha 1-protease inhibitor secreted by HepG2 cells.

Interleukin-6 (IL-6) induces changes in gene expression and the N-glycosylation pattern of acute-phase proteins in hepatocytes. IL-6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL-6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80-derived soluble human IL-6-receptor (shIL-6-R) significantly enhanced the IL-6 effect on N-glycosylation changes (revealed by reactivity with the lectin-concanavalin A) of a1-protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL-6-cDNA into HepG2 cells (HepG2-IL-6) resulting in constitutive secretion of 2 micrograms of IL-6 per 10(6) cells in 24 h led to a down-regulation of surface-bound gp80 and subsequent homologous desensitization of HepG2-IL-6 cells towards IL-6. Soluble human IL-6-R functionally substituted membrane-bound gp80 resulting in a reconstitution of responsiveness of HepG2-IL-6 cells.     

Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: Implications for PET dopamine studies

Affinities of dopamine (DA) analogs to both granular and plasma membrane uptake transporters were measured in vitro by inhibition of [³H]DA uptake in bovine chromaffin granule ghosts and C6 glial cells transfected with cDNA for the rat presynaptic dopamine transporter, respectively. Five amines were studied: DA, 6-fluorodopamine (6FDA), m-tyramine (MTA), 6-fluoro-m-tyramine (6FMTA), and β-fluoromethylene-m-tyramine (FMMTA). Direct uptake of ¹⁸F labeled 6FDA and 6FMTA was also measured in the chromaffin granule system and compared with [³H]DA uptake. Results show that the transporter affinities of 6FDA and MTA were similar to that of DA in both transport systems while affinities of 6FMTA and FMMTA were lower. Furthermore while the direct uptake of DA and FDA in chromaffin granules were essentially identical and significantly reserpine-inhibitable, the direct uptake of 6FMTA was about 15-fold less and only minimally sensitive to reserpine pretreatment. Thus, although vesicular protection and reuptake may influence the turnover of FDA in 6-fluoroDOPA studies, they are unlikely to be important determinants of the kinetics of the slowly clearing components in studies with either 6-fluoro-m-tyrosine (6FMT) or 6-fluoro-β- fluoro-methylene-m-tyrosine (6FFMMT), the bioprecursors of 6FMTA and 6-fluoro-FMMTA, respectively. These results are consistent with the finding that the longterm component in 6FMT PET studies is 6-fluoro-hydroxyphenylacetic acid (6FHPAC), which can be explained by the lack of vesicular protection of 6FMTA from MAO oxidation.     

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose: the chemical synthesis of isotopically labeled 2-epi-5-epi-valiolone analogs

2-epi-5-epi-valiolone is a cyclization product of the C(7) sugar phosphate, sedoheptulose 7-phosphate, involved in the biosynthesis of the aminocyclitol moieties of acarbose, validamycin, and pyralomicin. As part of our investigation into the pathway from 2-epi-5-epi-valiolone to the valienamine moiety of acarbose, we prepared 1-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-6], 5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-17], 1-epi-2-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-12] and 2-epi-5-epi-(6-(2)H(2))valiolamine [(6-(2)H(2))-11]. Compounds (6-(2)H(2))-6 and (6-(2)H(2))-17 were synthesized from 2,3,4,6-tetra-O-benzyl-D-glucopyranose in 10 and seven steps, respectively, whereas (6-(2)H(2))-12 and (6-(2)H(2))-11 were synthesized from 2,3,4,6-tetra-O-benzyl-D-mannopyranose in eight and 10 steps, respectively.