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Biomedical subjects

N M Farber

Publications and source records attributed to N M Farber.

7 recordsLinked to original sources
PubMed1987

cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells.

Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambda gt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that hybrid selection of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approximately 3.6 and approximately 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

Cell Line↗
PubMed1986

Identification of the interleukin-3 receptor using an iodinatable, cleavable, photoreactive cross-linking agent.

Murine interleukin-3 (mIL-3) is a lymphokine that stimulates the proliferation and differentiation of both pluripotent hemopoietic stem cells and their committed progeny. However, very little is known about the mechanism by which this growth factor elicits its effects on responsive cell populations. To gain insight into early events following mIL-3 receptor interaction, we initiated studies to isolate the receptor and study its properties. In this report, we demonstrate the use of a new iodinatable, cleavable, photoreactive cross-linking agent, sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate to identify the mIL-3 receptor. These studies reveal the mIL-3 receptor to be a single polypeptide chain with a molecular weight of 67 kDa and an isoelectric point of approximately 6.2.

Animals↗
PubMed1986

Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells.

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.

Amino Acid Sequence↗
PubMed1986

Inability of autoimmune mice with the lpr gene to spontaneously produce interleukin 3.

A recent report (Palacios, R., Eur. J. Immunol. 1984. 14: 599) indicates that spleen cells from MRL-MpJ-lpr/lpr mice spontaneously produce interleukin 3 (IL 3) and suggests that this lymphokine could be responsible for promoting the lymphoid hyperplasia regulated by the lpr gene. The present study shows that splenocytes from several murine strains bearing the lpr gene do not spontaneously produce IL3. This has been determined by measuring the amount of this lymphokine by a bioassay and by the detection of IL3 mRNA using Northern blot analysis. We suggest that another lymphokine is responsible for growth of lymphocytes regulated by the lpr gene.

Animals↗
PubMed1986

Purification of human erythropoietin to homogeneity by a rapid five-step procedure.

Human urinary erythropoietin (Ep) has been purified using a simple five-step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.

Amino Acid Sequence↗