Viral interleukin 6 stimulates human peripheral blood B cells that are unresponsive to human interleukin 6.

Cellular responsiveness to human interleukin 6 (hIL6) requires the expression of two receptor molecules: IL6-specific receptor (CD126'IL6R') and a nonspecific signal-transducing molecule (CD130'gp130'). Regulation of responsiveness to hIL6 is generally controlled by CD126'IL6R' expression. A viral homologue of hIL6 (vIL6) is encoded by human herpesvirus-8 and has biologic activity similar to hIL6 on a number of cell lines. vIL6 differs from hIL6 in its receptor utilization, requiring only CD130'gp130'. Total human B cells isolated from peripheral blood, which are predominantly CD126'IL6R'-negative, as well as sorted CD126'IL6R'-negative B cells, could be stimulated by recombinant vIL6, but not by hIL6, as indicated by induction of IL6-like signaling (STAT3 phosphorylation). This suggests that the ability of vIL6 to stimulate B cells expressing little or no CD126'IL6R' allows it to act on a larger pool of target B cells, compared to human IL6.     

Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.     

Cutting edge: soluble IL-6R is produced by IL-6R ectodomain shedding in activated CD4 T cells

IL-6 trans-signaling via the soluble IL-6R (sIL-6R) plays an important role in the progression of several autoimmune diseases and cancer by providing IL-6-responsiveness to cells lacking IL-6R. However, the potential sources of sIL-6R are less understood. In this study we show that sIL-6R is produced by both naive and memory CD4 T cells upon TCR activation. The production of sIL-6R by activated CD4 T cells is mediated by shedding of the membrane-bound IL-6R, and this process correlates with the expression of the metalloproteinase ADAM17 in these cells. In contrast to CD4 T cells, CD8 T cells do not express ADAM17 and their production of sIL-6R is negligible. Thus, during an immune response CD4 T cells are an important source of sIL-6R. Production of sIL-6R by autoreactive CD4 T cells may contribute to their role in the development of autoimmune disease by conferring IL-6-responsiveness to cells lacking IL-6R such as synoviocytes.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

人IL-6 /sIL-6R 结合分子模型的建立及在药物筛选中的应用

目的：建立人IL-6 /sIL-6R 结合的分子模型,用于筛选IL-6 /sIL-6R的抑制剂。方法：将人IL-6基因克隆至原核表达载体pET28a（+）中表达IL-6蛋白,western blot及人IL-6检测试剂盒分析鉴定表达蛋白。同法将人sIL-6R在pET15b载体中表达,纯化并用western blot检测目的蛋白。依据ELISA原理建立IL-6 /sIL-6R 结合的分子模型,并通过改变IL-6、sIL-6R及IL-6 antibody的浓度来优化该模型,用于IL-6 /sIL-6R拮抗药物的筛选。结果：人IL-6可在载体PET28a（+）中高效表达,且经western blot鉴定正确,人IL-6检测试剂盒检测显示具有较高的免疫活性。sIL-6R在PET15b中表达,western blot鉴定正确。通过对IL-6 /sIL-6R结合的分子模型的优化,得到其最佳条件为：IL-6R 1?g/well, IL-6 500ng/well, IL-6 antibody 1?g/well。应用该模型筛选发现有些化合物可显著抑制IL-6与其受体的结合。结论：成功构建IL-6 /sIL-6R结合的分子模型,为高通量筛选IL-6拮抗剂提供平台。     

Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K)

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.     

Enhancement of interleukin 6 cytostatic effect on human breast carcinoma cells by soluble IL-6 receptor from urine and reversion by monoclonal antibody.

Interleukin 6 receptor soluble urinary protein (IL-6-R-SUP), a purified urinary protein binding IL-6 and identified as a truncated 50 kDa soluble form of the 80 kDa IL-6 cellular receptor, was tested for its biological activity. Addition of IL-6-R-SUP enhances the growth stimulation of mouse plasmacytoma T1165 by subliminal concentrations of human recombinant IL-6. Since this effect could be due to a lower affinity of human IL-6 for the mouse cell receptor, we tested the effect of IL-6-R-SUP on human cells. We show that the growth-inhibitory effect of IL-6 on breast carcinoma cells is enhanced by addition of IL-6-R-SUP to these human cells although they possess abundant IL-6 receptors. With IL-6-R-SUP, complete growth inhibition by IL-6 could be achieved and the cells became more sensitive to low levels of IL-6. These effects were prevented by a monoclonal antibody against IL-6-R-SUP which blocks IL-6 binding to cells. The naturally occurring IL-6-R-SUP may help to increase the growth-regulatory action of IL-6.     

Cyclic acetals of 4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose and their conversion into methyl ether derivatives

The acid-catalysed reaction of 4,1',6'-trichloro-4,1',6'-trideoxy-galacto- sucrose (1) with 5.5 equiv. of 2-methoxypropene in N,N-dimethylformamide followed by acetylation gave 3',4'-di-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene-6-O-(1-methoxy-1-methylethyl)-galacto-sucrose (2, 2%), 6,3',4'- tri- O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O-isopropylidene-galacto -sucrose (3, 31%), 3',4'-di-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene- galacto-sucrose (4, 38%), 3'-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene- galacto-sucrose (5, 13%), and 2,3',4'-tri-O-acetyl-4,1',6'-trichloro- 4,1',6'-trideoxy-galacto-sucrose (6, 13%). Methylation of 4 followed by removal of the protecting groups gave 4,1',6'-trichloro-4,1',6'-trideoxy-6-O-methyl- galacto- sucrose (8). 4,1',6'-Trichloro-4,1',6'-trideoxy-3-O-methyl-galacto-sucrose (11) was synthesised from 6 by preferential tert-butyldiphenylsilylation of HO-6 followed by methylation and removal of the protecting groups. Likewise, 4,1',6'-trichloro- 4,1',6'-trideoxy-4'-O-methyl-galacto-sucrose (14) was synthesised from 5. Treatment of 3 with aqueous acetic acid followed by methylation and removal of the protecting groups afforded 4,1',6'-trichloro-4,1'6'-trideoxy-2,3-di-O-methyl- galacto-sucrose (17).     

Abnormal segregation of prion protein octapeptide-repeat alleles in cattle

The study was conducted on full-families of Black-and-White cattle obtained as 25 AI sire families and 355 cows, as well as their progenies, mostly heifers at the age of 1-3 months. The sire group was composed by the casual qualification of 10 PRNP 6/6 and 15 PRNP 6/5 individuals on the basis of accessible young progenies. The randomly selected group of cows is characterised by a very high frequency of PRNP 6/6 (74.9%), followed by lower frequency of PRNP 6/5 (24.5%) and a very low frequency of PRNP 5/5 genotype (0.6%). The progenies represent all expected genotypes, such as: PRNP 6/6 (60.5%), PRNP 6/5 (35.8%) and PRNP 5/5 (3.7%), respectively. Taking into consideration the genotypes of parents and progenies, the segregation of PRNP 6 and PRNP 5 alleles was analysed. Results of the non-informative mating variant of male symbol PRNP 6/6 x female symbol PRNP 6/6 (n = 87) are affected by the PRNP 6/6 progeny genotype in all cases. Subsequently, the results of mating variants male symbol PRNP 6/6 x female symbol PRNP 6/5 (n = 29) and male symbol PRNP 6/5 female symbol PRNP 6/6 (n = 179) showed statistically non-significant differences in both above-mentioned alternations. The progeny group related from male symbol PRNP 6/5 x female symbol PRNP 6/5 parental mating obtained fully informative and most valuable results based on the presented research concept. In the common group of 58 calves, the genotype PRNP 6/6 is represented by 26 individuals (44.8%), PRNP 6/5 - by 19 individuals (32.8%) and PRNP 5/5 - by 13 individuals (22.4%). Therefore, the theoretical genotype rate (25% : 50% : 25%) is drastically deformed and the differentiation between the observed and expected numbers of animals is statistically highly significant (chi(2) = 12.72; 2 df.). These differences are affected by two times higher PRNP 6/6 homozygous (chi(2) = 9.12; 1 df.) and responsively by the low number of PRNP 6/5 heterozygous animals (chi(2) = 3.45; 1 df.). Further investigations are carried out to explain the genetic determination of abnormal PRNP octa-peptide repeat allele segregation, which suggests possible lethal cis-trans linkage effects.     

Localization of Phosphoglucose Isomerase in Escherichia coli and Its Relation to the Induction of the Hexose Phosphate Transport System

The localization of phosphoglucose isomerase (PGI) was studied in relation to the induction of hexose phosphate uptake in Escherichia coli. The uptake system is induced only by extracellular glucose-6-phosphate (G6P); there is no induction by intracellular G6P. Fructose-6-phosphate (F6P) is an indirect inducer, and isomerization of F6P to G6P must occur before induction. PGI has been considered to be an internal enzyme; therefore, uptake of F6P by noninduced cells and leakage of the G6P formed would be required for induction. In this study, it was concluded that part of the PGI activity is located in the cell surface because: (i) uninduced, intact cells are able to convert F6P to G6P, whereas the activity of G6P dehydrogenase is not detectable; (ii) when cells are subjected to osmotic shock, about 10% of the PGI activity is found in the shock fluid; and (iii) sorbitol-6-phosphate (S6P) inhibits both PGI activity of whole cells and the induction of hexose phosphate transport system by F6P. S6P was not taken by intact cells. The data indicate that the isomerization of F6P to G6P can take place on the cell surface, and this explains the indirect induction of hexose phosphate transport by F6P.     

Regulation of ribosomal S6 kinase 2 by effectors of the phosphoinositide 3-kinase pathway

Ribosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5'-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitro and is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase Czeta but that S6K2 is more sensitive to basal activation by myristoylated protein kinase Czeta than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases.     

A novel retinoid binding property of human annexin A6

Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.     

Normal platelets possess the soluble form of IL-6 receptor

Interleukin 6 is a multifunctional cytokine that exerts its biological activity through binding to an 80 Kd specific receptor (IL-6Ralpha) and a 130 Kd signal-transducing unit (gp130). A 55 Kd soluble IL-6R (IL-6sR) has also been described which, after binding to IL-6 is also able to activate gp130. The presence of IL-6Ralpha was described in some megakaryoblastic cell lines but is still controversial in normal megakaryocytes. In this study we demonstrate the presence of intraplatelet IL-6sR by Western blot through the appearance of a 55 Kd protein and the finding of detectable amounts of IL-6sR in the platelet content by ELISA technique. Besides, we showed IL-6sR release during platelet activation induced by thrombin and a complex of ADP and epinephrine. IL-6Ralpha on platelet membrane could not be found neither by Western blot nor by flow cytometry. The IL-6sR released during platelet activation and complexed to IL-6 could act on cell types such as endothelial cells that do not possess IL-6Ralpha through binding to gp130.Besides, since we could not find IL-6R on platelet membrane, the potentiating effect of IL-6 on platelet function could be explained through binding of IL-6sR/IL-6 complex to platelet membrane gp130.     

Accumulation of porphobilinogen and other pyrroles by mutant and wild type Rhodopseudomonas spheroides: regulation by heme

Certain mutant strains of Rhodopseudomonas spheroides accumulate coproporphyrin(ogen) and porphobilinogen when incubated with low aeration in malate-glutamate medium supplemented with glycine and succinate. Strains 6-6 and 6-6R have barely detectable levels of uroporphyrinogen synthetase and accumulate porphobilinogen. Strain 6-6R is more active than 6-6 in porphobilinogen formation but is less active in heme synthesis. Production of porphobilinogen by strain 6-6 is stimulated by addition of δ-aminolevulinate or o-phenanthroline but neither additive affects strain 6-6R. Strain 2–33 accumulates porphobilinogen and coproporphyrin(ogen) and is exceptionally low in heme synthesis. Inhibition of bacteriochlorophyll synthesis by puromycin in the wild type and strain 6-6R is accompanied by an acceleration in heme synthesis. The wild type accumulates coproporphyrin(ogen) upon addition of o-phenanthroline but this is prevented by the prior addition of puromycin. It is concluded that the excess production of pyrroles by the mutants and by the wild type in response to o-phenanthroline is attributable to failure of feedback control of δ-aminolevulinate synthetase by heme. The mutants are convenient sources of porphobilinogen and coproporphyrin.     

Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6

Ribosomal protein S6 is phosphorylated in response to mitogens by activation of one or more protein kinase cascades. Phosphorylation of S6 in vivo is catalyzed by (at least) two distinct mitogen-activated S6 kinase families distinguishable by size, the 70 kDa and 90 kDa S6 kinases. Both S6 kinases are activated by serine/threonine phosphorylation. Members of each family have been cloned. The 90 kDa S6 kinases are activated more rapidly than the 80 kDa S6 kinase, and may have other intracellular targets. The 70 kDa S6 kinase is relatively specific for 40 S ribosomal subunits. No kinase capable of activating the 70 kDa S6 kinase has been identified. Members of the 90 kDa S6 kinases are activated in vitro by 42 kDa and 44 kDa MAP kinases, which are in turn activated by mitogen-dependent activators. The pathways for mitogen-stimulated S6 phosphorylation are discussed.