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R Hewick

Publications and source records attributed to R Hewick.

9 recordsLinked to original sources
PubMed1999

A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos.

The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of an inhibitor of haemopoietic stem cell proliferation.

The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.

Amino Acid Sequence↗
PubMed1989

Characterization and NH2-terminal amino acid sequence of natural human interleukin for DA cells: leukemia inhibitory factor. Differentiation inhibitory activity secreted by a T lymphoma cell line.

Six human T lymphomas and an NK-like cell line were tested for their ability to produce HILDA, one of the two human growth promoting activities for the DA1.a cells. Among them, the HSB2 cell line turned out to be the only one secreting significant HILDA activity (200-400 units/ml) after activation with 50 nM phorbol myristate acetate. Subclones of the HSB2 cell line were obtained by limiting dilution experiments. One of them (2B3) was found to secrete 1,000-5,000 units/ml of HILDA after phorbol myristate acetate activation in the presence of 10% fetal calf serum and 200-500 units/ml in serum-free conditions. 2B3-HILDA was purified from serum-free conditioned medium by a four-step procedure including fast flow cationic exchange at pH 6, concanavalin A chromatography, reverse-phase high performance liquid chromatography and gel-filtration high performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified radiolabeled cytokine revealed a single band of Mr 43,000, which co-electrophoresed with the biological activity. The NH2-terminal amino acid sequence of the first residues of the protein were determined and found to be similar to the equivalent residues deduced from the molecularly cloned cytokine. Isoelectric point determination revealed some charge heterogeneity of HILDA, which focused to pH 8.5-9 after neuraminidase treatment. Carbohydrate content of the cytokine was studied by deglycosylation experiments which showed that the O-linked oligosaccharides represented 2,000-3,000 and that the N-linked sugars account for half of the apparent molecular weight of HILDA.

Amino Acid Sequence↗
PubMed1985

Regulatory region of the heat shock-inducible capR (lon) gene: DNA and protein sequences.

The CapR protein is an ATP hydrolysis-dependent protease as well as a DNA-stimulated ATPase and a nucleic acid-binding protein. The sequences of the 5' end of the capR (lon) gene DNA and N-terminal end of the CapR protein were determined. The sequence of DNA that specifies the N-terminal portion of the CapR protein was identified by comparing the amino acid sequence of the CapR protein with the sequence predicted from the DNA. The DNA and protein sequences established that the mature protein is not processed from a precursor form. No sequence corresponding to an SOS box was found in the 5' sequence of DNA. There were sequences that corresponded to a putative -35 and -10 region for RNA polymerase binding. The capR (lon) gene was recently identified as one of 17 heat shock genes in Escherichia coli that are positively regulated by the product of the htpR gene. A comparison of the 5' DNA region of the capR gene with that of several other heat shock genes revealed possible consensus sequences.

Amino Acid Sequence↗
PubMed1984

Characterization of Factor V activation intermediates.

Bovine Factor V was partially activated with bovine beta-thrombin and activation intermediates, and end products were isolated either by column chromatography under nondenaturing conditions or electroelution from slab gels following electrophoresis in dodecyl sulfate or both. Electrophoresis of partially activated single-chain Factor V (Mr = 330,000) revealed intermediates designated B (Mr congruent to 205,000) and C (Mr congruent to 150,000), plus end products designated C1 (Mr congruent to 120,000), D (Mr congruent to 94,000), E (Mr congruent to 74,000), F (Mr congruent to 71,000), and G (Mr congruent to 31,000). All components except C1 were visualized readily by staining with Coomassie blue. C1, however, did not stain with this dye but was readily visualized with the Schiff-periodate or silver staining procedures. Chromatography of samples of partially activated Factor V on QAE (quaternary aminoethyl)-cellulose in Ca2+ yielded fractions consisting, respectively, of B plus C and B plus D. Both were biologically active, but the former pair required further exposure to thrombin to yield activity, whereas the latter did not. Either pair, when treated with EDTA, lost activity and could be resolved by further chromatography on QAE-cellulose into isolated components B, C, and D. Activity was recovered upon reconstitution of the pairs in Ca2+, suggesting that B plus C or B plus D comprise two subunit proteins, both subunits of which are required for biological activity. Gel electrophoretic analysis of isolated B and C, after further exposure to thrombin, indicated that B is the precursor of end products C1, E, and G, whereas C is the precursor of D and F. Conventional NH2-terminal sequence analysis and amino acid composition analysis indicated that C and D share the same NH2 terminus with Factor V and that the weighted composition of B plus C is the same as Factor V. Microsequence analysis of C1 and E indicated that C1 has the NH2-terminal sequence of intermediate B, which differs from that of end product E. These results indicate that: 1) a single cleavage of Factor V yields B and C from the COOH and NH2 termini, respectively, of the parent; 2) the pair B and C constitute a Ca2+-stabilized protein of two subunits, both of which are required for subsequent expression of biological activity; 3) the precursor-product relationship between B and C and the end products of activation are: B----C1 + E + G, and C----D + F; and 4) end product D is derived from the NH2 terminus of C, and end product C1 is derived from the NH2 terminus of B.

Amino Acid Sequence↗
PubMed1983

N-terminal amino acid sequences of the alpha and beta chains of HLA-DR1 and HLA-DR2 antigens.

The N-terminal amino acid sequences of the alpha and beta chains of HLA-DR1 and HLA-DR2 antigens were obtained with subnanomole quantities of material by using a gas-liquid solid-phase sequencer. A comparison of the N-terminal amino acid sequences of HLA-DR1 and HLA-DR2 alpha chains revealed no differences. However, in the first 35 N-terminal residues of the beta chains from HLA-DR1 and HLA-DR2 antigens, two regions of variability are readily apparent, each comprising about six amino acids. Conceivably one or both of these variability regions may be responsible for the serologically defined polymorphism of HLA-DR alloantigens.

Amino Acid Sequence↗
PubMed1981

Microchemical instrumentation.

A number of cell surface molecules of great theoretical and practical importance simply cannot be obtained in amounts sufficient for molecular analysis using conventional methods and instrumentation. Because of our interest in such studies, we began about eight years ago to explore the possibility of developing new instrumentation for the sequence analysis of very small quantities of polypeptide chains. These efforts have led to the development of two microsequenators which employ one thousandth to one ten-thousandth the material used in the original sequenator described by Per Edman. In addition, in conjunction with the explosion of recombinant DNA techniques, we also have begun to develop instrumentation for the sequence analysis and synthesis of DNA molecules. In this paper we describe briefly several new instruments that have been developed at Caltech. We believe this new instrumentation in conjunction with the recombinant DNA and hybridoma technologies will provide unique opportunities to analyze cell-surface molecule in the years ahead.

Amino Acid Sequence↗
PubMedDate not supplied

Isolation and characterization of genomic and cDNA clones of human erythropoietin.

The glycoprotein hormone erythropoietin regulates the level of oxygen in the blood by modulating the number of circulating erythrocytes, and is produced in the kidney or liver of adult and the liver of fetal or neonatal mammals. Neither the precise cell types that produce erythropoietin nor the mechanisms by which the same or different cells measure the circulating oxygen concentration and consequently regulate erythropoietin production are known. Cells responsive to erythropoietin have been identified in the adult bone marrow, fetal liver or adult spleen. In cultures of erythropoietic progenitors, erythropoietin stimulates proliferation and differentiation to more mature red blood cells. Detailed molecular studies have been hampered, however, by the impurity and heterogeneity of target cell populations and the difficulty of obtaining significant quantities of the purified hormone. Highly purified erythropoietin may be useful in the treatment of various forms of anaemia, particularly in chronic renal failure. Here we describe the cloning of the human erythropoietin gene and the expression of an erythropoietin cDNA clone in a transient mammalian expression system to yield a secreted product with biological activity.

Amino Acid Sequence↗