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C Sprecher

Publications and source records attributed to C Sprecher.

12 recordsLinked to original sources
PubMed2002

Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors.

OBJECTIVE: The aim of this study was to examine the effects of recombinant human Fgf18 on chondrocyte proliferation and matrix production in vivo and in vitro. In addition, the expressions of Fgf18 and Fgf receptors (Fgfr) in adult human articular cartilage were examined. METHODS: Adenovirus-mediated transfer of Fgf18 into murine pinnae and addition of FGF18 to primary cultures of adult articular chondrocytes were used to assess the effects of FGF18 on chondrocytes. In situ hybridization was used to examine the expression of Fgf18 and Fgfr s in adult human articular cartilage. RESULTS: Expression of Fgf18 by adenovirus-mediated gene transfer in murine pinnae resulted in a significant increase in chondrocyte number. Chondrocytes were identified by staining with toluidine blue and a monoclonal antibody directed against type II collagen. Fgf18, Fgfr 2-(IIIc), Fgfr 3-(IIIc), and Fgfr 4 mRNAs were detected within these cells by in situ hybridization. The nuclei of the chondrocytes stained with antibodies to PCNA and FGF receptor (FGFR) 2. Addition of FGF18 to the culture media of primary articular chondrocytes increased the proliferation of these cells and increased their production of extracellular matrix. To assess the receptor selectivity of FGF18, BaF3 cells stably expressing the genes for the major splice variants of Fgfr1-3 were used. Proliferation of cells expressing Fgfr 3-(IIIc) or Fgfr 2-(IIIc) was increased by incubation with FGF18. Using FGFR-Fc fusion proteins and BaF3 cells expressing Fgfr 3-(IIIc), only FGFR 3-(IIIc)-Fc, FGFR 2-(IIIc)-Fc or FGFR 4-Fc reduced FGF18-mediated cell proliferation. Expression of Fgf18, Fgfr 3-(IIIc) and Fgfr 2-(IIIc) mRNAs was localized to chondrocytes of human articular cartilage by in situ hybridization. CONCLUSION: These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro. Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage.

Adult↗
PubMed2001

STR primer concordance study.

Over 1500 population database samples comprising African Americans, Caucasians, Hispanics, Native Americans, Chamorros and Filipinos were typed using the PowerPlex 16 and the Profiler Plus/COfiler kits. Except for the D8S1179 locus in Chamorros and Filipinos from Guam, there were eight examples in which a typing difference due to allele dropout was observed. At the D8S1179 locus in the population samples from Guam, there were 13 examples of allele dropout observed when using the Profiler Plus kit. The data support that the primers used in the PowerPlex 16, Profiler Plus, and COfiler kits are reliable for typing reference samples that are for use in CODIS. In addition, allele frequency databases have been established for the STR loci Penta D and Penta E. Both loci are highly polymorphic.

Alleles↗
PubMed2001

Interleukin 20: discovery, receptor identification, and role in epidermal function.

A structural, profile-based algorithm was used to identify interleukin 20 (IL-20), a novel IL-10 homolog. Chromosomal localization of IL-20 led to the discovery of an IL-10 family cytokine cluster. Overexpression of IL-20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL-20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL-20 receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL-20, a novel cytokine identified solely by bioinformatics analysis.

Animals↗
PubMed2000

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

Amino Acid Sequence↗
PubMed1994

Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: mechanism of action with trypsin and factor Xa as model proteinases.

The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.

Amino Acid Sequence↗
PubMed1994

Expression, purification and characterization of a Kunitz-type protease inhibitor domain from human amyloid precursor protein homolog.

The Kunitz-type protease inhibitor domain from a recently identified homolog of the Alzheimer amyloid precursor protein (APPH KPI) was expressed in yeast, purified and characterized. Its inhibition profile towards several serine proteases was studied and compared to that of APP KPI, the Kunitz domain from the Alzheimer amyloid precursor protein. APPH KPI was shown to inhibit proteases with trypsin-like specificity with an inhibitor profile resembling that of the APP KPI domain. The KPI domains from APP and APPH inhibited trypsin (Ki = 0.02 nM), and plasma kallikrein (Ki = 86 nM) with approximal equal affinity. In comparison to APP KPI (Ki = 82 nM) the KPI domain of the homolog, APPH KPI, (Ki = 8.8 nM) was a more potent inhibitor of glandular kallikrein. APPH KPI was a less potent inhibitor of chymotrypsin than APP KPI (Ki = 78 nM as compared to Ki = 6 nM), plasmin (Ki = 81 nM as compared to 42 nM), and factor XIa (Ki = 14 nM as compared to Ki = 0.7 nM). The affinity of factor XIa for APPH KPI is sufficiently high to allow for an interaction in the blood. It is, however, well possible that the physiological protease ligand for the receptor-like APPH protein has yet to be identified.

Amino Acid Sequence↗
PubMed1991

Expression of recombinant human plasminogen in mammalian cells is augmented by suppression of plasmin activity.

We present evidence that over-expression of human plasminogen, the precursor to the serine protease plasmin, can be cytotoxic to mammalian cells. When an expression vector containing plasminogen cDNA is transfected into baby hamster kidney cells, the number of drug-resistant colonies as well as the levels of plasminogen secreted by those colonies is lower than observed in similar transfections of other protease precursor genes. The recombinant plasminogen accumulates intracellularly as degraded NH2-terminal fragments. In contrast, a mutant of plasminogen that produces inactive plasmin (active site Ser740 changed to Ala) is synthesized by these cells as a full-length plasminogen molecule, and the colony numbers and expression levels are normal. Thus, the generation of plasmin activity is responsible for the cytotoxic phenomena and the degradation associated with plasminogen expression. In addition, experiments using a plasminogen mutant that cannot be activated to plasmin (activation cleavage site Arg560 to Gly) or using coexpression of antisense urokinase RNA indicate that an endogenous plasminogen activator is responsible for converting newly synthesized plasminogen to plasmin. Finally, coexpression of plasminogen with alpha 2-plasmin inhibitor, a serpin which is the physiologic inhibitor of plasmin, prevents the toxic effects of intracellular plasmin activity and allows the synthesis and secretion of native human plasminogen.

Animals↗
PubMed1987

Expression of mouse histone genes: transcription into 3' intergenic DNA and cryptic processing sites downstream from the 3' end of the H3 gene.

Introduction of the mouse histone H3.1 gene into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene resulted in the production of two mRNAs from the transfected gene, one with a normal 3' end and the other one with a longer 3'-untranslated region, ending at site X, which was poly(A)+. In contrast, the endogenous histone H3.1 gene only produced a single mRNA. The cryptic poly(A)+ site was only used when the histone H3.1 gene was transfected. To localize possible downstream cryptic processing sites, the hairpin loop at the end of the histone gene was deleted and the resulting deletions were introduced into L cells. Two major mRNAs were produced from this gene, one ending at site X and the major one ending at site Y, which was located 150 nucleotides before site X. Transcription extended downstream of site X efficiently in the endogenous gene, as judged by the extent of transcription of downstream sequences in isolated nuclei. Transcription extended downstream of site X in the transfected gene because the placement of a normal histone 3' end downstream of site X resulted in transcripts that ended at site X and longer transcripts that ended with the new histone 3' end. These results indicate that transcription may normally proceed a substantial distance past the hairpin loop (greater than 500 bases). The formation of the different 3' ends in these transfected genes was due to competition between different processing mechanisms.

Animals↗
PubMed1985

Structure of the sea urchin U1 RNA repeat.

The genes coding for U1 RNA in the sea urchin L. variegatus are present in a 1400 base pair tandem repeat. One member of the repeat has been cloned and its sequence determined. The repeat unit contains a single copy of the gene for L. variegatus U1 RNA. This gene encodes an RNA which is 75% homologous to mammalian U1 RNA. The L. variegatus U1 RNA could assume a secondary structure similar to that proposed for other U1 RNAs. In addition the L. variegatus U1 RNA is precipitated by anti-SM and anti-RNP antisera. Analysis of the L. variegatus genomic DNA using the cloned U1 gene as a probe reveals a major and a minor type of repeat unit. The two repeated units are the same length but differ in a number of restriction enzyme sites clustered 200-500 bases down-stream from the gene. The monomer we have cloned and sequenced is a representative of the minor repeat. A sequence (GATAA) which is -41 to -37 bases 5' to the gene has homology to the putative RNA polymerase II promoter. Fifteen bases 3' of the gene is a sequence (CAAAGAAAGAAAA) which is very similar to the sequence found 3' of the sea urchin histone genes. The two Hha I, Hpa II and Ava I sites in the repeat are all unmethylated in sperm DNA.

Animals↗
PubMed1985

Regulated expression of a chimeric histone gene introduced into mouse fibroblasts.

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

Animals↗