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R A Kastelein

Publications and source records attributed to R A Kastelein.

At least 37 records · Page 2Linked to original sources
PubMed1999

IL-18 and CD28 use distinct molecular mechanisms to enhance NK cell production of IL-12-induced IFN-gamma.

NK cells play an important role in innate immune resistance, particularly through synthesis of the pro-inflammatory cytokine IFN-gamma. This study compares the abilities of the cytokine IL-18 and the costimulatory cell surface molecule CD28 to enhance IL-12-driven IFN-gamma production by NK cells. Studies with other cytokines (IL-1beta, IL-6, TNF-alpha, IL-15) showed that IL-18 or anti-CD28 treatments were the most efficient inducers of IFN-gamma when combined with IL-12. The ability of IL-18 to enhance IFN-gamma was shown to be dependent on the presence of IL-12. Similarly, although anti-CD28 stimulation alone could enhance IFN-gamma synthesis, this effect was significantly increased in the presence of IL-12. Although neither method of costimulation required de novo protein synthesis for their effects on IFN-gamma mRNA expression, these molecules used distinct mechanisms. Specifically, nuclear run-on analysis revealed that IL-18 in combination with IL-12 enhanced the rate of transcription of the IFN-gamma gene. Conversely, treatment with anti-CD28 plus IL-12 did not significantly up-regulate the rate of transcription of the IFN-gamma gene, but stabilized IFN-gamma mRNA expression within NK cells. These findings illustrate costimulatory pathways that result in potent IFN-gamma responses by NK cells and show that although IL-18 and anti-CD28 can enhance the synthesis of IL-12-driven IFN-gamma, they employ molecular mechanisms that are distinct from one another.

Animals↗
PubMed1999

IL-10 enhances NK cell proliferation, cytotoxicity and production of IFN-gamma when combined with IL-18.

Previous studies have shown that IL-10 inhibits the accessory cell functions required for production of IFN-gamma by T cells and NK cells. Our results show that although IL-10 did not induce the production of IFN-gamma by NK cells, it did enhance the ability of IL-18 to stimulate NK cell production of IFN-gamma. In addition, IL-10 augmented NK cell proliferation and cytotoxic activity when combined with IL-18. However, IL-10 did not affect the ability of IL-12 to stimulate NK cells to produce IFN-gamma or proliferate, but there was an additive effect with IL-12 to increase NK cell cytotoxic activity. Interestingly, the type I IFN, whose receptors (R) are related to the IL-10R, also enhanced the effects of IL-18 on NK cell production of IFN-gamma and NK cell cytotoxicity. The ability of IL-10 to elevate the production of IFN-gamma appeared to be specific for NK cells since IL-10 had no effect on the production of IFN-gamma by Th1 clones stimulated with IL-18 or IL-12 in the presence of a monoclonal antibody specific for CD3. These latter results correlated with lower mRNA levels for the alpha and beta chains of the IL-10R in Th1 cells than observed in NK cells. Thus, the ability of IL-10 and IL-18 to up-regulate NK cell function, but not Th1 cell activity, appears to be based on expression of the IL-10R.

Adjuvants, Immunologic↗
PubMed1999

Inclusion of cetaceans within the order Artiodactyla based on phylogenetic analysis of pancreatic ribonuclease genes.

Mammalian secretory ribonucleases (RNases 1) form a family of extensively studied homologous proteins that were already used for phylogenetic analyses at the protein sequence level previously. In this paper we report the determination of six ribonuclease gene sequences of Artiodactyla and two of Cetacea. These sequences have been used with ruminant homologues in phylogenetic analyses that supported a group including hippopotamus and toothed whales, a group of ruminant pancreatic and brain-type ribonucleases, and a group of tylopod sequences containing the Arabian camel pancreatic ribonuclease gene and Arabian and Bactrian camel and alpaca RNase 1 genes of unknown function. In all analyses the pig was the first diverging artiodactyl. This DNA-based tree is compatible to published trees derived from a number of other genes. The differences to those trees obtained with ribonuclease protein sequences can be explained by the influence of convergence of pancreatic RNases from hippopotamus, camel, and ruminants and by taking into account the information from third codon positions in the DNA-based analyses. The evolution of sequence features of ribonucleases such as the distribution of positively charged amino acids and of potential glycosylation sites is described with regard to increased double-stranded RNA cleavage that is observed in several cetacean and artiodactyl RNases which may have no role in ruminant or ruminant-like digestion.

Amino Acid Sequence↗
PubMed1999

Target detection by an echolocating harbor porpoise (Phocoena phocoena).

Two echolocation experiments are described. They were conducted on the same harbor porpoise housed in a sea pen, one year apart at Neeltje Jans, The Netherlands. The aims were to determine the target detection ability of an echolocating harbor porpoise, with the ultimate goal to predict the distance at which harbor porpoises can detect fishing nets. In experiment 1, the maximum distance at which the 3-year-old porpoise could detect a 7.62-cm diameter water-filled stainless-steel sphere by echolocation was determined psychophysically. The 50%-current detection threshold was reached when the sphere was at a distance of 26 m from the porpoise's rostrum. In experiment 2, conducted a year later, the maximum detection distance for a 5.08-cm water-filled stainless-steel sphere was 15.9 m. The target strengths of both targets were measured using simulated harbor porpoise echolocation signals and the results, coupled with transmission-loss calculations, indicated that the echo levels received by the porpoise with the targets at the threshold ranges in the two experiments were only 1.3 dB apart. Together with information on the target strengths of various fishing nets, the results of the present study can be used to predict the distance at which the nets can be detected by harbor porpoises.

Animals↗
PubMed1999

Transmission beam pattern and echolocation signals of a harbor porpoise (Phocoena phocoena).

The transmission beam pattern of an echolocating harbor porpoise (Phocoena phocoena) was measured in both the vertical and horizontal planes. An array of seven Brüel and Kjaer 8103 hydrophones connected to an amplifier-line driver module was used to measure the beam patterns. The porpoise was trained to station in a hoop and echolocate a cylindrical target located at a range between 7 and 9 m while the array was located 2 m in front of the hoop. The 3-dB beamwidth in both the vertical and horizontal planes was the same at approximately 16 degrees and the beam was pointed toward the forward direction. The individual hydrophones in both the vertical and horizontal arrays measured signal waveforms that were similar throughout the 40-degree span of the array. The porpoise emitted signals with intervals that were 20 to 35 ms longer than the round trip travel time between the animal and the target. The average source level, peak frequency, and bandwidth were 157 dB, 128 kHz, and 16 kHz, respectively.

Amplifiers, Electronic↗
PubMed1998

A family of human receptors structurally related to Drosophila Toll.

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors--named TLRs 1-5--are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role--akin to Toll in the dorsoventralization of the Drosophila embryo--as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, we also show that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel beta/alpha fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.

Amino Acid Sequence↗
PubMed1997

Genetic structure and chromosomal mapping of MyD88.

The myeloid differentiation (MyD) marker MyD88 was initially characterized as a primary response gene, upregulated in mouse M1 myeloleukemic cells in response to differentiation induced by interleukin-6. Subsequent analysis revealed that MyD88 possesses a unique modular structure, which consists of an N-terminal "death domain," similar to the intracellular segments of TNF receptor 1 and Fas, and a C-terminal region related to the cytoplasmic domains of the Drosophila morphogen Toll and vertebrate interleukin-1 receptors. In this report we describe the cloning and gene structure of mouse MyD88. The complete coding sequence of mouse MyD88 spans five exons, with the first exon encoding the complete death domain. Zooblot analysis revealed that MyD88 is an evolutionarily conserved gene. MyD88 was localized to the distal region of mouse chromosome 9 by interspecific backcross mapping. The human homolog (hMyD88) was mapped to chromosome 3p22-p21.3 by PCR analysis of a human chromosome 3 somatic cell hybrid mapping panel. Northern blot analysis revealed widespread expression of MyD88 in many adult mouse tissues, and RT-PCR studies detected MyD88 mRNA in T and B cell lines and differentiating embryonic stem cells. The broad expression pattern demonstrates that mouse MyD88 expression is not restricted to cells of myeloid lineage as was originally believed.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Comparison of the effects of interleukin-1 alpha, interleukin-1 beta and interferon-gamma-inducing factor on the production of interferon-gamma by natural killer.

Interferon-gamma inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-gamma (IFN-gamma) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1 gamma. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1 alpha, IL-1 beta and IGIF on NK cell production of IFN-gamma. All three IL-1 types enhanced NK cell production of IFN-gamma when induced by IL-2 or IL-12, although at high concentrations (> 10 ng/ml), IGIF was five- to tenfold more potent than IL-1 alpha or IL-1 beta. This effect correlated with enhanced levels of mRNA for IFN-gamma when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-gamma was not increased by IL-1 alpha or IL-1 beta. The ability of IGIF to enhance IFN-gamma production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-gamma and demonstrate that the effect of IGIF on NK cell production of IFN-gamma is similar to that of IL-1 alpha and IL-1 beta but distinct from that of IL-12.

Adjuvants, Immunologic↗
PubMed1997

Isolation, characterization and chromosomal localization of human WNT10B.

The Wnt family constitutes a set of structurally related glycoproteins that have been implicated in oncogenesis and developmental processes. The vertebrate Wnt10 family includes mouse, Xenopus and salamander proteins. We undertook a bioinformatics based approach to characterize a novel human Wnt gene. The gene (WNT10B) encodes a 389-amino acid protein with 96.6% sequence identity to mouse Wnt10b. The expression pattern of WNT10B reveals that it is synthesized in many adult tissues with the highest levels found in heart and skeletal muscle. WNT10B expression was also detected in several human cancer cell lines with elevated mRNA levels observed in HeLa (cervical cancer) cells. WNT10B was localized to 12q13.1 by PCR typing of a human/rodent monochromosomal panel and fluorescent in situ hybridization.

Adult↗
PubMed1996

Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism?

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.

Amino Acid Sequence↗
PubMed1996

Molecular characterization and modular analysis of human MyD88.

MyD88 was first characterized as a myeloid differentiation primary response gene in mice, activated in M1 myeloleukemic cells following interleukin-6 (IL-6) induced growth arrest and terminal differentiation. Analysis of expressed sequence tags (ESTs) from activated dendritic cell libraries led to the indentification of cDNAs encoding the human homolog (hMyD88). The original description of MyD88 as a 243 aa protein may reflect a truncated mouse cDNA since the 2682 nt hMyD88 cDNA predicts a 296 aa cytoplasmic protein. Consistent with this proposal is the detection of a 33 kDa protein in human heart, kidney and liver tissue. The expression pattern of MyD88 is also more widespread than originally believed: a 2.6 kb hMyD88 mRNA species was found to be constitutively expressed in many adult human tissues; in addition MyD88 expression was observed in monocyte, T, B, NK and dendritic cells. The MyD88 protein has a modular structure composed of an N-terminal 'death domain' (DD) similar to the intracellular segments of TNF receptor 1 (TNFR1) and FAS and a C-terminal region related to the signaling domains of vertebrate interleukin-1 receptors (IL-1R) and the Drosophila morphogen Toll. This intriguing structural framework may endow MyD88 with unique signaling capabilities.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Functional replacement of cytokine receptor extracellular domains by leucine zippers.

Granulocyte-macrophage colony-stimulating factor receptor signals by a complex which includes the ligand and two different receptor subunits: a low affinity alpha receptor binding chain (granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-Ralpha)) and a signal-transducing beta chain (GM-Rbeta). To investigate two unresolved issues in the initiation of signaling, the role of receptor extracellular domains and receptor stoichiometry, we replaced the mouse GM-Ralpha and GM-Rbeta extracellular domains with the leucine zipper domain of either the Fos or Jun molecule. We co-transfected combinations of chimeric receptors into Ba/F3 cells and found that both simple heterodimers of the GM-Ralpha and GM-Rbeta intracellular domains and homodimers of the GM-Rbeta intracellular domain signaled for proliferation. Surprisingly, homodimers of the GM-Ralpha intracellular domain also signaled for prevention of apoptosis in transfected cells. We similarly engineered dimers of the intracellular domain of the human interferon gamma receptor beta subunit and found that homodimers of the intracellular domain signaled for proliferation. When Fos peptide was added to Ba/F3 cells expressing the human interferon gamma receptor beta subunit construct, thereby preventing homodimer formation, the cells no longer proliferated in the absence of mouse interleukin 3.

Amino Acid Sequence↗
PubMed1996

The leptin haemopoietic cytokine fold is stabilized by an intrachain disulfide bond.

Structure prediction algorithms have tagged leptin as the newest member of the haemopoietic cytokine family, a diverse class of secreted hormone-like factors with pleiotropic effects in immunity and haemopoietic development. While haemopoietic cytokines typically lack sequence similarity, they conserve a distinctive three-dimensional fold, a four-alpha-helix bundle structure that is recognized by the cognate family of haemopoietic cellular receptors. We have constructed a detailed molecular model of the human leptin helical fold that places the two cysteine residues of the leptin chain, Cys96 and Cys146, in close spatial proximity to each other. In this report, we present evidence that these cysteines are involved in an intrachain disulfide bridge that is critical for the structural integrity and stability of leptin. A leptin variant that is unable to form the disulfide link shows a reduced biological response when administered to leptin-deficient, ob/ob mice.

Algorithms↗
PubMed1996

Mouse IL-17: a cytokine preferentially expressed by alpha beta TCR + CD4-CD8-T cells.

A novel cytokine originally designated murine CTLA-8 was described as a cDNA isolated from an activated T cell hybridoma produced by fusing a mouse cytotoxic T cell clone and a rat T lymphoma. This cDNA, which contains mRNA instability sequences characteristic of many cytokines, encoded a putative secreted protein that was homologous to the ORF13 gene of Herpesvirus saimiri. The human homolog to this molecule has recently been identified as the proinflammatory cytokine IL-17. We describe the isolation of a cDNA encoding mouse IL-17 from a cDNA library generated from alpha beta TCR + CD4-CD8- thymocytes using a subtraction technique that enriched for activation specific genes. This cDNA shares 87.3% amino acid identity to the previously described murine CTLA-8. Comparison of murine CTLA-8 to a cDNA we isolated from activated rat splenocytes revealed that murine CTLA-8 is, in fact, the rat homolog of IL-17. Mouse IL-17 mRNA is specifically expressed by activated alpha beta TCR + CD4-CD8- T cells, a small subset with a potentially important role in immune regulation. Mouse, rat, and human IL-17 can induce IL-6 secretion in mouse stromal cells, indicating that all homologs can recognize the mouse receptor.

Animals↗
PubMed1995

Rational design of a mouse granulocyte macrophage-colony-stimulating factor receptor antagonist.

Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions at residues in the first alpha-helix were examined for biological activity and receptor binding properties. Substitution at the buried residue His15 affected both bioactivity and receptor binding. Of the four surface-exposed positions examined (Arg11, Lys14, Lys20, and Glu21) only substitutions at Glu21 impaired bioactivity. Proteins with charge reversal substitutions at this position were partial agonists and weak antagonists of native mGM-CSF action. All substitutions at Glu21 abrogated high affinity binding. Lys14 and Lys20 substitution proteins showed various receptor binding defects. Qualitative and quantitative measurement of these binding defects identified Lys14 as a residue that interacts specifically with the beta subunit of the mGM-CSF receptor, whereas Lys20 appeared to exist at the GM-R alpha-subunit/GM-R beta-subunit interface as substitutions at this position produce both high and low affinity binding losses. These determinations permitted the design of a more potent mGM-CSF antagonist.

Animals↗
PubMed1995

Expression and purification of a synthetic human obese gene product.

Human obese (hOB) protein was recently identified as a secreted hormone-like factor that is exclusively produced by adipose tissue and appears to regulate the size of the body's fat stores. We describe a rapid and efficient repetitive extension PCR method for the construction of a 453-bp synthetic hOB gene with high-frequency codons to optimize expression in Escherichia coli. The use of a bacterial signal sequence fused to hOB together with expression of bacteriocin release protein resulted in efficient release of hOB into the culture medium. The protein was readily purified to homogeneity from the culture medium using a two-step procedure.

Amino Acid Sequence↗