Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae.

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5'-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

Ciliary neurotrophic factor induces acute-phase protein expression in hepatocytes.

During inflammatory states, hepatocytes are induced to synthesize and secrete a group of proteins called acute-phase proteins. It has recently been shown that besides interleukin-6 (IL-6), related cytokines such as leukemia inhibitory factor, oncostation M and interleukin-11 are also mediators of the hepatic acute-phase response. All these mediators belong to the hematopoietic family of alpha-helical cytokines. Here we show that an additional member of this cytokine family, ciliary neurotrophic factor (CNTF), induces the hepatic acute-phase protein genes haptoglobin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and beta-fibrinogen in human hepatoma cells (HepG2) and in primary rat hepatocytes with a time course and dose-response comparable with that of IL-6. Our next aim was to define the receptor components used by CNTF on hepatic cells. Using a cell-free binding assay we exclude that CNTF binds to the 80 kDa IL-6 receptor, a protein with significant homology to the CNTF receptor which has recently been cloned from neuroblastoma cells. In human hepatoma cells (Hep3B) which lack the leukemia inhibitory factor receptor, CNTF was not able to induce acute-phase protein synthesis, indicating that this receptor protein may be part of the functional CNTF receptor on hepatic cells.

Alternative assay procedures for cytokines and soluble receptors of the IL-6 family.

Human hepatoma cells (HepG2 cells) were transfected with expression vectors for human IL-6 (hIL-6) and rat IL-6R (rIL-6-R). The cell lines were used for testing the biological activity of different IL-6 species, soluble hIL-6R (shIL-6R) and some members of the IL-6 cytokine family by means of an ELISA procedure. The assay is based on induction of the gene expression of the acute phase protein haptoglobin in hepatoma cells and provides an alternative bioassay taking advantage of the hepatocyte stimulatory activity of IL-6 (as opposed to the B9 proliferative assay). A dose-response experiment with IL-6 showed that half-maximal stimulation was achieved with approx. 5 ng/ml of hIL-6 in HepG2 cells and with 5-10 ng/ml muIL-6 in HepG2-rIL-6R cells after 24 h. The same response was achieved with 10 ng/ml shIL-6R in HepG2-IL6 cells. In conclusion, the assay is fast and reliable and might be adopted for other cytokines and receptors with hepatocyte stimulating activity.

Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus.

The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70-80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius. The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein.

The centromere-binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae.

A gene encoding the centromere binding factor 1 (Cbf1p) of the human pathogenic yeast Candida albicans was cloned and characterized. An open reading-frame was detected which encoded a 223 amino acid protein with a calculated molecular weight of 25.8 kDa and a relative isoelectric point of 5.55. It shares 39% overall amino acid sequence identity with Saccharomyces cerevisiae Cbf1p. We localized the CaCBF1 gene on chromosome 4. Southern analysis indicated that CaCBF1 is probably present as a single copy gene per haploid genome. The CaCBF1 gene under the control of its own promoter was able to complement the methionine auxotrophic growth, the increased mitotic instability of CEN plasmids and the slow growth of a Saccharomyces cerevisiae cbf1Delta mutant strain.

Sorption-catalytic determination of cadmium using bromobenzothiazo noncovalently bound to silica and paper.

Cadmium (along with Fe(II), Co(II), Zn(II), and Pb(II) ions) decreases the rate of oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with KIO4 conducted either without or with Mn(II) as a catalyst. Cadmium(II) is preconcentrated from aqueous solutions on silica plates or paper filters physically modified with a reagent for selective determination of Cd(II), namely 1-[(6-bromo-2-benzothiazolyl)azo]-2-naphthol (bromobenzothiazo, or BBT). The modifier is strongly retained on the both supports at pH 6-10 and does not affect the inhibiting effect of Cd(II) in the indicator reaction. Cadmium is determined by its inhibiting action directly on the sorbents by measuring transmittance (BBT/paper) or reflectance (BBT/silica) with limits of detection of 2 x 10(-4) and 0.03 mg/L, respectively. The proposed hybrid combination of sorption with catalytic detection on the sorbent allows to increase the selectivity factors several times (up to 2 orders) relatively to the determination in solution. Tap water samples and soil extracts were analyzed.

Sorption-catalytic determination of manganese directly on a paper-based chelating sorbent.

A hybrid technique based on a catalytic reaction carried out on the surface of a paper-based sorbent is proposed. It is shown that MnII exhibits its catalytic action in the oxidation of 3,3',5,5'-tetramethylbenzidine with periodate in aqueous solution as well as on filter-paper with or without attached diethylenetriaminetetraacetate (DETATA) groups. Optimum conditions differ for the reaction in solution and on filter-paper. For equal catalyst concentrations, higher initial reaction rates are attainable on the filter-papers. Preconcentration of manganese on the DETATA sorbent combined with the subsequent catalytic reaction improves the selectivity, reduces the limit of determination down to 5 x 10(-6) mg l-1 (as compared with 6 x 10(-5) mg l-1 in solution) and expands the linear range by an order of magnitude (to 5 x 10(-6)-2.5 x 10(-3) mg l-1). The precision of manganese determination on the sorbent is high (the RSDs are < or = 5% for > or = 6 x 10(-4) micrograms Mn). Samples of tap and river water were analyzed by use of the proposed sorption-catalytic technique. A rapid procedure for the determination of manganese with visual detection was also developed.

Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor.

An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-alpha-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.

Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata.

The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C. glabrata partial genomic library. Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20.9 kDa. The amino acid sequence is highly homologous (78.6% identity) to ScCbf3d and 48.3% identical to the human homologue p19 (SKP1). Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene. The cloned CgCBF3D gene can functionally substitute the S. cerevisiae homologue in a S. cerevisiae CBF3D-deletion mutant. The GenBank Accession No. for this gene is AF 072472.

Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1.

The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Structural and functional studies on the human hepatic interleukin-6 receptor. Molecular cloning and overexpression in HepG2 cells.

cDNAs coding for the human hepatic interleukin-6 receptor (IL-6-R) have been isolated from a library made from poly(A) RNA of dexamethasone-treated human hepatoma cells (HepG2). We found the hepatic IL-6-R to be identical to the one expressed by leucocytes. A polyclonal antiserum was raised in rabbits against the IL-6-R protein expressed in Escherichia coli. Although the entire IL-6-R protein was used for immunization, only antibodies to the cytoplasmic domain of the IL-6-R were obtained. It is demonstrated by affinity cross-linking and subsequent immunoprecipitation with antibodies against the ligand as well as against the receptor that the cloned cDNA codes for the functional IL-6-R on HepG2 cells. When the hepatic IL-6-R cDNA was overexpressed in HepG2 cells, these cells became more sensitive to low concentrations of IL-6 with respect to the induction of gamma-fibrinogen mRNA.

Biosynthesis and half-life of the interleukin-6 receptor and its signal transducer gp130.

Interleukin-6 (IL-6) exerts its action via a receptor complex composed of a ligand-binding subunit (gp80) and a signal transducer (gp130) which both belong to the hematopoietic receptor super-family. Very little is known about the biosynthesis and the biological half-lives of proteins of this superfamily. Therefore, we studied the biosynthesis and maturation of the interleukin-6 receptor and its signaling subunit gp130 by pulse chase experiments in stably transfected Madin-Darby canine kidney cells. We found that both proteins are synthesized as precursors with apparent molecular masses of 67 kDa and 130 kDa, respectively. These receptor forms are processed within 45-60 min into mature proteins of 82 kDa and 150 kDa containing complex-type oligosaccharides. The signal transducer gp130 shows a similar maturation in human hepatoma cells HepG2. The IL-6 receptor appears at the cell surface 45 min after completion of its synthesis in the endoplasmic reticulum. In both cell types studied, gp80 and gp130 are rapidly turned over with half-lives of 2-3 h. These half-lives were unaffected by the presence of the ligand IL-6.

Identification of a region within the cytoplasmic domain of the interleukin-6 (IL-6) signal transducer gp130 important for ligand-induced endocytosis of the IL-6 receptor.

Interleukin-6 (IL-6) exerts its action via a cell surface receptor complex consisting of two subunits, the IL-6 receptor and the signal transducer gp130. We have studied the role of both transmembrane proteins for IL-6 internalization and ligand-induced down-regulation of cell surface receptors. Co-expression of wild-type and mutant forms of both the IL-6 receptor and gp130 in transiently transfected COS-7 cells revealed that gp130 is essential for efficient endocytosis and receptor down-regulation. Whereas the cytoplasmic domain of the IL-6 receptor is not significantly involved in the internalization process, deletion of the corresponding domain of gp130 resulted in an almost complete loss of the ability to endocytose IL-6. Mutants with different truncations within the intracellular domain of gp130 revealed that a 10-amino acid sequence TQPLLDSEER is crucial for efficient internalization. Since this sequence contains a putative di-leucine internalization motif, we suggest that a di-leucine motif directs the receptor mediated endocytosis of the IL-6 receptor complex.

The hepatic interleukin-6 receptor. Studies on its structure and regulation by phorbol 12-myristate 13-acetate-dexamethasone.

Recombinant human 125I-interleukin-6 (IL-6) was cross-linked with the homobifunctional reagent disuccinimidyl suberate to human hepatoma cells (HepG2). Three recombinant human 125I-IL-6-containing complexes of apparent molecular masses of 100, 120, and 200 kDa were immunoprecipitated with specific antibodies to human IL-6 or to the 80-kDa IL-6 receptor subunit. We show by immunoprecipitation, peptide mapping, and by the use of a cleavable heterobifunctional cross-linker (Denny-Jaffe reagent) that different polypeptides are involved in the formation of the 100- and 120-kDa IL-6-containing complexes. The molecular compositions of the 100- and 120-kDa cross-linked complexes were identified. The 100-kDa complex consisted of one ligand and one IL-6 receptor subunit, glycoprotein 80 (gp80), whereas the 120-kDa complex was found to be composed of one ligand and a polypeptide which was immunoprecipitable with the monoclonal antibody AM64 directed against gp130. Exposure of HepG2 cells to phorbol 12-myristate 13-acetate (PMA) or PMA-dexamethasone led to an increase in the 80-kDa IL-6 receptor mRNA and functional receptor protein. Whereas treatment of HepG2 cells with PMA led to an increase in the formation of gp80.gp130.IL-6 complexes determined by cross-linking, no corresponding increase in high affinity binding sites was found. The existence of a third IL-6 receptor subunit present in limiting amounts on HepG2 cells is proposed to explain this discrepancy. Evidence is presented that the 80-kDa IL-6 receptor up-regulation by PMA-dexamethasone is caused by the depletion of protein kinase C since the protein kinase C inhibitor staurosporine mimics the effect of PMA-dexamethasone.

Recombinant soluble human interleukin-6 receptor. Expression in Escherichia coli, renaturation and purification.

The recombinant soluble human interleukin-6 receptor (srhIL-6R) was expressed in Escherichia coli as a non-glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL-6R could be renaturated by an in vitro folding procedure using L-arginine and the glutathione-redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL-6-Sepharose column. The functional features of the recombinant soluble receptor were further analysed. A part of the extracellular domain (amino acids 145-345) of the human interleukin-6 receptor (IL-6R) was expressed in E. coli and the purified protein was used to raise antibodies in rabbits. Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies. Since the antiserum obtained did not interfere with IL-6 binding, it could be used to establish a cell-free IL-6-binding assay, In this assay, the srhIL-6R bound IL-6 with an affinity of Kd = 1.5 nM as measured by Scatchard-plot analysis. When 125I-IL-6 was chemically cross-linked to the purified srhIL-6R and analyzed by SDS/PAGE, several 125I-IL-6-containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL-6/IL-6R complex. The srhIL-6R was shown to exhibit biological activity, i.e. it stimulated acute-phase protein synthesis in the recently established human hepatoma cell line HepG2-IL-6 which does not express the IL-6-binding subunit of the IL-6R complex on the cell surface.

Sympathetic neurons can produce and respond to interleukin 6.

Neuronal expression of cytokines is an area of active investigation in the contexts of development, disease, and normal neural function. Although cultured rat sympathetic neurons respond very weakly to exogenous interleukin 6 (IL-6), we find that addition of soluble IL-6 receptor (sIL-6R) and IL-6 enhances neuronal survival in the absence of nerve growth factor. Neutralizing monoclonal antibodies against IL-6 block these effects. Addition of IL-6 and sIL-6R also induces a subset of neuropeptide and transmitter synthetic enzyme mRNAs identical to that demonstrated for leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Both of these effects are duplicated by addition of a highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide chain, which is designated H-IL-6. In addition, we show that sympathetic neurons produce IL-6. In situ hybridization indicates a neuronal localization of IL-6 mRNA in superior cervical ganglia, and bioactive IL-6 protein is detected in ganglion culture supernatants. Interestingly, the IL-6 produced by sympathetic neurons does not lead to survival of these cells in culture unless sIL-6R is added. Thus, sympathetic neurons can produce IL-6 and may respond to it in an autocrine/paracrine manner if sIL-6R is present. Moreover, the prior findings of sIL-6R in serum and inflammatory fluids now have added interest in the context of neuro-immune interactions.

The soluble interleukin-6 receptor is generated by shedding.

The ligand-binding subunit (gp80) of the human interleukin-6 receptor (IL-6R) was transiently expressed in COS-7 cells. The metabolically labeled protein was shown to be quantitatively released from the membrane within 20 h. We identified the protein released from the transfected COS-7 cells after purification to homogeneity and N-terminal sequencing as a soluble form of the gp80/IL-6R. Shedding of the gp80 protein was strongly induced by 4 beta-phorbol-12-myristate-13-acetate, indicating that the process was regulated by protein kinase C (PKC). This was further corroborated by the finding that co-transfection of a PKC expression plasmid led to enhanced shedding of the gp80 protein. Since shedding of gp80 could not be prevented by treatment of the cells with inhibitors of all known classes of proteases, a novel protease seems to be involved. As a control, an unrelated membrane protein (vesicular stomatitis virus glycoprotein) was transfected into COS-7 cells and analyzed for shedding. Since the turnover of this protein was not mediated by shedding, we conclude that the release of gp80 from COS-7 cells is a specific process. The shed gp80 protein specifically binds IL-6, and this complex shows biological activity on human hepatoma cells. Human peripheral blood monocytes released a soluble form of the gp80 protein into the culture medium upon PMA treatment indicating that PKC-regulated shedding is the physiological mechanism of generation of the soluble IL-6R.

Structure-function analysis of interleukin-6 utilizing human/murine chimeric molecules. Involvement of two separate domains in receptor binding.

As an approach to understanding the interaction of interleukin-6 (IL-6) and its 80-kDa receptor (gp80), we have constructed chimeric human/murine IL-6-molecules, which were expressed in Escherichia coli and analyzed for biological activity and receptor binding. This experimental strategy was based on the observation that human IL-6 acts on human and murine cells, whereas murine IL-6 stimulates only murine cells. The regions to be exchanged were chosen according to the four antiparallel helix model of the hematopoietic cytokine family. All 14 chimeras constructed showed biological activity on murine cells. From the differential biological activities on human cells we deduced that three out of four domains of IL-6 are involved in species specificity, whereas only two domains are necessary for specific recognition by the gp80 IL-6-receptor protein.