Biomedical subjects

E M Alderman

Publications and source records attributed to E M Alderman.

14 recordsLinked to original sources

PubMed1992

Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3.

IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to it receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs of these mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor.

Adult↗

PubMed1992

The pediatrician's role in the diagnosis and treatment of substance abuse.

Abuse of drugs and alcohol is pervasive in our society. The role of the pediatrician as a health-care provider from birth through young adulthood includes recognizing the stigmata in newborns of prenatally ingested drugs as well as being able to treat neonatal drug withdrawal syndromes. Questioning about drug use and other closely related topics should be incorporated into all health-care maintenance visits, starting at the age of 10 y, with parental participation and anticipatory guidance being offered. Physical examination and laboratory testing are not as helpful in confirming an impression of substance abuse as a comprehensive interview and a full appreciation of the warning signs of substance abuse. Treatment of the problematic user with a multidisciplinary team that understands adolescent development and behavior as well as the problem of substance abuse is crucial. The decision to treat the teen in an ambulatory or inpatient setting is determined by the extent of abuse, underlying medical problems and psychopathology, and the degree of family dysfunction. The pediatrician must not avoid addressing these issues with patients. If, however, upon identifying an adolescent or a newborn with a drug problem, the pediatrician feels uncomfortable or ill-prepared to manage the patient, appropriate professional referrals are warranted. Despite exercising the referral option for treatment, as advocate for child and family, the pediatrician remains professionally bound to track all drug abuse-related referrals while continuing to participate in the general ongoing care of the patient and family. Finally, the pediatrician should retain a public advocacy role in the community by offering educational, preventive, and supportive efforts in the continuing struggle against drug abuse.

Adolescent↗

PubMed1991

An internal ribosome binding site can be used to select for homologous recombinants at an immunoglobulin heavy-chain locus.

The encephalomyocarditis virus (EMCV) leader sequence is responsible for efficient, cap-independent translation initiation from the viral RNA. It has been used to increase the expression of internal coding regions on polycistronic mRNA encoded by recombinant DNA constructs. We have designed a sequence-replacement-type vector for targeting to immunoglobulin heavy-chain loci in hybridoma cells. Homologous recombination of this vector introduces a human gamma 1 constant-region sequence linked to the EMCV leader and a neomycin phosphotransferase (neo) gene. The resulting cells express a bicistronic mRNA encoding at the 5' end a chimeric murine VDJH-human C gamma 1 heavy chain, followed by neo linked to the internal ribosome binding site provided by the EMCV leader. These homologous recombinants express the chimeric heavy chain at levels equivalent to the heavy chain in the parental hybridoma. This strategy of using an EMCV-neo cassette to obtain efficient selectable marker gene expression has potential application to a range of gene targeting vectors.

Animals↗

PubMed1990

High level synthesis of immunoglobulins in Chinese hamster ovary cells.

The expression of lambda L and microH chain cDNA was examined in Chinese hamster ovary cells. Each cDNA was linked to a different, amplifiable, selectable drug marker gene, and expression was monitored in the presence of increasing concentrations of the selective drugs. Cells were obtained that produced greater than 60 micrograms/10(6) cells/48 h of assembled antibody. This Chinese hamster ovary cell-synthesized IgM was polymeric, and exhibited specific hapten binding and C fixation. The expression strategy employed here may prove useful for the future production of genetically engineered antibodies and other multi-subunit proteins.

Animals↗

PubMed1990

Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies.

We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N-terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO-dependent cell line. Only Group II MoAbs inhibit a newly developed EPO-dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

Animals↗

PubMed1986

Immunocytochemical and enzymatic detection of lysozyme in human colon carcinoma cell lines.

Six of a total of 14 human colon carcinoma cell lines produce and secrete lysozyme in vitro. Three also produce the enzyme when propagated in vivo in athymic mice. None of the lysozyme positive cells stained in a manner typical of Paneth cells. Additionally, lysozymes from all six colon lines possess identical molecular weights (approximately 14,000 daltons).

Animals↗

PubMed1985

Angiogenic activity of human tumor plasma membrane components.

Plasma membranes from the human colon adenocarcinoma cell line HT-29 have been isolated and examined for the presence of angiogenic activity. Membrane-associated macromolecules extracted with Triton X-100 were fractionated on immobilized wheat germ agglutinin. The fraction which bound specifically (about 200 ng of protein/mL packed cells) was highly angiogenic when assayed on the chick embryo chorioallantoic membrane. As little as 0.2 ng of this human tumor derived material consistently induced neovascularization. Similarly, 1-2 ng of this material implanted into the rabbit cornea induced new vessel growth (5-8 mm) within 10 days. The plasma membranes of eight other human tumor lines were examined for angiogenic activity. For each, the wheat germ agglutinin bound material induced neovascularization at the low nanogram level. In contrast, the wheat germ agglutinin bound material derived from purified plasma membranes of two normal human diploid fibroblast cell lines failed to induce an angiogenic response on the chick chorioallantoic membrane, even at microgram levels.

Animals↗

PubMed1985

Partial purification and characterization of a vascular permeability factor secreted by a human colon adenocarcinoma cell line.

The established human colon adenocarcinoma cell line, HT-29, secretes a vascular permeability factor (VPF) in vitro. The factor has been purified from serum-free conditioned medium by acidification, cation-exchange, and reverse-phase and anion-exchange high-performance liquid chromatography. The VPF is a non-glycosylated acidic protein of apparent molecular weight 45,000, and is at least 4 orders of magnitude more potent than histamine as an inducer of vascular permeability. Its biological activity is unaffected by soybean trypsin inhibitor or by inhibitors of histamine, kinins, prostaglandins, or acid proteases. The VPF induces vascular permeability within minutes, which suggests a direct effect on the endothelial cell.

Adenocarcinoma↗

PubMed1985

Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells.

The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

Adenocarcinoma↗

PubMed1985

Amino acid sequence of human tumor derived angiogenin.

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMed1985

Induction of angiogenesis by bovine brain derived class 1 heparin-binding growth factor.

The angiogenic capacity of the class 1 heparin-binding growth factor from bovine brain, an anionic endothelial cell mitogen of Mr 16 000, has been evaluated. Its ability to induce the growth of new blood vessels has been assessed by means of two established assay systems. On the embryonic chick chorioallantoic membrane dose-response studies demonstrate that 160 ng (10 pmol) of mitogen is required to induce angiogenesis in greater than 50% of the eggs within 72 h. In the presence of 1 unit of exogenous heparin only 40 ng of mitogen (2.5 pmol) is needed to induce a similar response. Moreover, this occurs within 48 h, indicating that heparin also augments the angiogenic response by enhancing the rate of induction of angiogenesis. Eighty nanograms (5 pmol) of mitogen also induces the ingrowth of new blood vessels into the rabbit cornea, both in the presence and in the absence of heparin. These results establish that the class 1 heparin-binding growth factor from bovine brain is an angiogenesis factor. Importantly, the neovascularization induced by this angiogenesis factor is enhanced by heparin. The mechanistic implications for neovascularization under certain normal and pathological conditions are discussed.

Animals↗

PubMed1985

Lysozyme: a major secretory product of a human colon carcinoma cell line.

One of the major proteins secreted by an established human colon adenocarcinoma cell line has been isolated in 25% yield from the serum-free medium in which the cells were grown and identified as lysozyme. Its purification was achieved by sequential steps of acidification, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography. It was recognized to be a human lysozyme on the basis of its molecular weight (14 000), isoelectric point (10.5), amino acid composition, and enzymatic activity. Its identity with previously characterized human lysozymes was established by amino-terminal sequence, peptide composition, immunological properties, NMR, and crystallography. A 4-day, 7-L collection of conditioned medium contained 20.3 mg of secreted protein of which 4.9 mg or approximately 24% of the total was tumor-derived lysozyme. The intracellular level of lysozyme was approximately 18 ng per 10(6) carcinoma cells. The possible significance of these findings in regard to the malignant process and tumor maintenance is discussed.

Adenocarcinoma↗

PubMed1985

Isolation of tumor-secreted products from human carcinoma cells maintained in a defined protein-free medium.

A protein-free synthetic cell-growth medium has been defined that permits long-term survival (greater than 120 days) of an established human colon tumor cell line, HT-29. Viability is dependent upon both the concentration of L-glutamine in the medium and the cell density at the time of initial transfer into it. Cell proliferation is minimal, thus obviating the necessity for subculturing. HT-29 adenocarcinoma cells maintained in large-scale culture with this medium continue to secrete the established colon tumor marker carcinoembryonic antigen as well as growth factors and lysozyme. These and, potentially, other important tumor-derived products can therefore be generated continuously in such cultures so that they can be isolated from a conditioned medium free of contaminating serum and protein supplements.

Carcinoembryonic Antigen↗

PubMed1979

Cyclic adenosine 3',5'-monophosphate regulation of the bacteriophage T6/colicin K receptor in Escherichia coli.

Mutant strains of Escherichia coli unable to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) or the cyclic adenosine monophosphate receptor protein (CRP) were more resistant than wild-type cells to infection by bacteriophage T6. This resistance was found to be associated with the decreased production of specific T6 receptor protein (also the colicin K receptor) located in the outer membrane protein fraction of these cells. Transcription of this particular outer membrane protein was regulated by the cAMP-CRP complex. A novel affinity technique coupled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used in these investigations.

Bacterial Proteins↗