Marked differences between two isoforms of human pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.     

Marked differences in differentiation propensity among human embryonic stem cell lines

The differentiation potential of 17 human embryonic stem (hES) cell lines was compared. Some lines exhibit a marked propensity to differentiate into specific lineages, often with >100-fold differences in lineage-specific gene expression. For example, HUES 8 is best for pancreatic differentiation and HUES 3 for cardiomyocyte generation. These non-trivial differences in developmental potential among hES cell lines point to the importance of screening and deriving lines for lineage-specific differentiation.     

Thrombopoietic activity of human interleukin-6

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK-01 cells cultured in the presence of human interleukin-1. The N-terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N-terminal region of human interleukin-6 (IL-6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL-6 by Western analysis using an anti-IL-6 antibody. Commercial recombinant human IL-6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin-3 and increased the number of peripheral platelets in mice in a dose-dependent manner. From these results, it is concluded that human IL-6 has thrombopoietic activity.     

Analysis of excitatory microcircuitry in the medial entorhinal cortex reveals cell-type-specific differences

Medial entorhinal cortex (MEC) plays an important role in physiological processes underlying navigation, learning, and memory. Excitatory cells in the different MEC layers project in a region-specific manner to the hippocampus. However, the intrinsic microcircuitry of the main excitatory cells in the superficial MEC layers is largely unknown. Using scanning photostimulation, we investigated the functional microcircuitry of two such cell types, stellate and pyramidal cells. We found cell-type-specific intralaminar and ascending interlaminar feedback inputs. The ascending interlaminar inputs display distinct organizational principles depending on the cell-type and its position within the superficial lamina: the spatial spread of inputs for stellate cells is narrower than for pyramidal cells, while inputs to pyramidal cells in layer 3, but not in layer 2, exhibit an asymmetric offset to the medial side of the cell's main axis. Differential laminar sources of excitatory inputs might contribute to the functional diversity of stellate and pyramidal cells.     

Preparation of functionally active recombinant human interleukin-6

The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.     

Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins.

Monoclonal antibodies directed against the glycoproteins of human respiratory syncytial virus were used in competitive enzyme-linked immunosorbent assays for topological mapping of epitopes. Whereas epitopes of the F glycoprotein could be ascribed to five nonoverlapping antigenic sites, anti-G antibodies recognized unique epitopes, many of whose competition profiles overlapped extensively. Variant viruses selected with a neutralizing (47F) anti-F antibody lost the binding for only 47F and 49F antibodies, which mapped in the same antigenic area. In contrast, viruses selected with an anti-G antibody lost the capacity to bind most of the anti-G antibodies, and their G protein was not recognized by an anti-virus antiserum, indicating major changes in the antigenic structure of the G molecule. Finally, we found great antigenic variation of the G protein among viral isolates. This occurred even within viruses of the same subtype with only limited divergence of amino acid sequence between strains. All of these data indicate marked differences in the antigenic organization of the G and F glycoproteins of respiratory syncytial virus; we discuss these differences in terms of the chemical structure of the glycoproteins.     

Internal deletions in human interleukin-6: structure-function analysis

By cDNA mutagenesis, we have constructed internal and C-terminal deletions (delta 21-51, delta 52-97, delta 97-104, delta 127-174, delta 97-184 and delta 134-184) in human interleukin-6 (hIL-6). All those deletion-carrying hIL-6 (delta hIL-6) proteins were then produced in Xenopus laevis oocytes and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results show that, at least in frog oocytes, the first potential N-glycosylation site (Asn45) is utilized exclusively. The IL-6 conformation of these deletion-carrying proteins has been studied by immunoprecipitation with two kinds of monoclonal antibodies (mAb's): mAb's that show preference towards denatured hIL-6, or conformation-specific mAb's. The binding pattern of these two series of mAb's indicated that the IL-6 conformation has been largely destroyed for four of our delta-proteins. Proteins delta 21-51 and delta 127-174 have kept a part of the IL-6 tertiary structure since they are still recognized by some conformation-specific mAb's. All of these delta hIL-6 proteins were inactive in the IL-6 hybridoma growth factor (HGF) assay and unable to inhibit the HGF activity of the recombinant human wild-type IL-6 (wt hIL-6). Moreover, the oocyte-synthesized delta hIL-6 (delta 21-51, delta 127-174, delta 97-184, delta 134-184) did not bind to the IL-6 receptor. Finally, we have produced two proteins with aa 29-33 or 97-104 substituted by corresponding murine IL-6 (mIL-6) sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

Ethnic differences in the distribution of interleukin-6 polymorphisms among three Brazilian ethnic groups

Polymorphisms in the interleukin-6 promoter region have been associated with diseases. In this study we investigated the -634G/C and -174G/C IL-6 promoter polymorphisms in three Brazilian ethnic groups. We verified that the allele frequencies of the two polymorphisms and haplotype frequencies varied significantly between the populations.     

On the multimeric nature of natural human interleukin-6

Natural human interleukin-6 (IL-6) characterized under completely denaturing conditions consists of a set of differentially modified phosphoglycoproteins of molecular mass in the range from 23 to 30 kDa ("25-kDa" O-glycosylated species and "30-kDa" O- and N-glycosylated species). The 25-kDa O-glycosylated IL-6 (which contains only Ser- or Thr-GalNAc-Gal-NeuNAc and thus should not bind wheat germ or lentil lectins) bound to and was eluted from a wheat germ lectin affinity column by GlcNAc and from a lentil lectin affinity column by methyl-alpha-D-Man suggesting that the 25-kDa IL-6 species formed heteromeric complexes with the N-glycosylated 30-kDa IL-6. In non-denaturing gels (0.2% Nonidet P-40-polyacrylamide gel electrophoresis (PAGE)), even under reducing conditions (15 mM dithiothreitol or 1 M beta-mercaptoethanol and heating), fibroblast-derived IL-6 migrated as a predominant complex of mass approximately 85 kDa and additional minor 45-65-kDa complexes. Little IL-6 was detected in the size range 23-30 kDa. Elution of the major 85-kDa complex and re-electrophoresis through sodium dodecyl sulfate-PAGE revealed that it represented a heteromeric aggregate of the 25- and 30-kDa IL-6 species; the 45-65-kDa complexes were largely composed of the 25-kDa protein. The bulk of fibroblast-derived IL-6 eluted in the size range 45-85 kDa from a Sephadex G-200 gel filtration column further indicating that fibroblast-derived IL-6 was largely multimeric even in dilute solutions. Functionally, the high molecular mass IL-6 fractions from the G-200 column were less active in the B9 hybridoma growth factor assay than the lower molecular mass fractions but appeared to be equally active in the Hep3B hepatocyte-stimulating factor assay. Taken together, the data indicate that natural human IL-6 exists as a multimeric aggregate with varying biological activity.     

Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin-6 by fibroblasts transfected with human interleukin-6-receptor cDNA

A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.     

Regulation of interleukin-6 receptor expression in human monocytes and hepatocytes

Human blood monocytes normally express the interleukin-6 receptor. Treatment of cultured monocytes with endotoxin, interleukin-1 beta, or interleukin-6 results in a decrease in interleukin-6 receptor mRNA levels. Glucocorticoids aso cause a drop in monocytic interleukin-6 receptor mRNA levels. We also found interleukin-6 receptor expression in cultured human hepatocytes, but in contrast to monocytes, where interleukin-6 receptor mRNA is presented by the ligand and by interleukin-1, treatment of hepatocytes with interleukin-6 or interleukin-1 resulted in increased interleukin-6 receptor mRNA levels. Induction of interleukin-6 receptor mRNA in hepatocytes was less pronounced when glucocorticoids were omitted from the culture medium. We conclude that during noninflammatory homeostasis, blood monocytes are involved in binding of trace amounts of circulating interleukin-6. During inflammatory events, the main target of interleukin-6 may be changed from the monocytic population not only to activated B-cells, but also to the hepatocytes.     

LPS induces interleukin-6 and interleukin-8 but not tumor necrosis factor-alpha in human adipocytes

Adipose tissue-derived cytokines are presumably involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. Here we studied the lipopolysaccharide (LPS)-induced expression dynamics of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 in human adipose tissue biopsies, in preadipocyte-derived adipocytes, and in mesenchymal stem cell (MSC)-derived adipocytes. TNFalpha, IL-6, IL-8 and IL-10 secretions by adipose tissue explants were increased 5.5-, 19.5-, 3.5- and 12.5-fold, respectively, by LPS (1 microg/mL) administration. Concordantly, IL-6 and IL-8 release was dose-dependently induced in MSC-derived adipocytes by LPS (>10 pg/mL). In contrast, TNFalpha and IL-10 remained undetectable even at the highest LPS dose (1 microg/mL) after 24h. In MSC- and preadipocyte-derived adipocytes, respectively, exposure to LPS evoked a weak and transient induction of TNFalpha mRNA whereas induction of IL-6 and IL-8 mRNA were pronounced and sustained for at least 24h. Basal glucose uptake, lipolysis and IL-6 mRNA were induced by exogenous TNFalpha (10 ng/mL) but not by IL-6 (10 ng/mL), IL-8 (100 ng/mL) and IL-10 (20 ng/mL). In this adipocyte model TNFalpha induces well known metabolic effects, but together with previous reports these data suggest that inflammation-induced TNFalpha may derive from non-adipocyte sources in adipose tissue, likely to be macrophages.     

The role of interleukin-6 and interleukin-6/interleukin-6 receptor-alpha complex in the pathogenesis of multiple myeloma

Multiple myeloma (MM) is a plasma-cell disorder in which malignant plasma cells accumulate in the bone marrow and usually produce a monoclonal immunoglobulin. Usual presenting features of overt MM include recurrent osteolytic lesions, bacterial infections, anemia and renal insufficiency. MM is responsible for about 1 percent of all cancer-related deaths in Western countries. Its epidemiologic pattern remains obscure, and its cause unknown [1]. The presence of somatic mutations within the immunoglobulin genes of myeloma cells indicate that the putative myeloma-cell precursors have been stimulated by antigens within germinal centers and are either memory B cells or migrating plasmablasts. Myeloma cells proliferate slowly in the bone marrow and display a weak apoptotic index in vivo [2]. This suggest that some defects in the apoptotic process could be involved in this neoplasia. Interleukin-6 (IL-6) is known to be an essential survival factor of myeloma cells and to protect them from apoptosis induced by different stimuli (e.g. dexamethasone, CD95, serum starvation, gamma-irradiation). More recently, important works have been devoted to the biology of the soluble form of the IL-6R alpha i.e., sIL-6R alpha. These works give IL-6/sIL-6R alpha complex an important role in the biology of IL-6. The purpose of the current review is to emphasize the role of this complex in the pathogenesis of MM.