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Biomedical subjects

E B Dowdle

Publications and source records attributed to E B Dowdle.

At least 19 recordsLinked to original sources
PubMed1996

Abnormal association between invariant chain and HLA class II alpha and beta chains in chronic lymphocytic leukemia.

We have previously shown that peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL) express increased amounts of the minor p35 form of class II invariant chain (Ii) relative to the major p33 form. In this report we demonstrate in Western blots that in CLL lymphocytes, but not in normal or Epstein-Barr virus-transformed normal lymphocytes, p35 and p33 Ii form sodium dodecyl sulfate (SDS)-resistant complexes with class II alpha and beta chains and that these complexes form an abnormally large proportion of the total class II molecules. Others have shown that stable SDS-resistant alpha-beta complexes are only formed upon binding of exogenous antigenic peptides for presentation at the cell surface. Large amounts of p35 Ii remaining in the endoplasmic reticulum and capable of forming stable complexes with alpha and beta chains could compete with endogenous antigenic peptides for available class II peptide binding sites. The presentation of endogenous tumor antigens would thus be prevented, leading to the escape of the CLL clone from immunological surveillance.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1994

Basic fibroblast growth factor antagonizes transforming growth factor beta-mediated erythroid differentiation in K562 cells.

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta 1 (TGF-beta) have both been shown to act on hematopoietic progenitor cells. bFGF is a hematopoietic cytokine that acts on progenitor cells in concert with other cytokines to promote their proliferation. TGF-beta induces erythroid differentiation in K562 cells. To determine whether bFGF might act on progenitor cells by antagonizing the effects of cytokines that induce differentiation, we determined the effects of bFGF on the TGF-beta-mediated induction of hemoglobin synthesis in K562 cells. bFGF antagonized the TGF-beta-mediated induction of hemoglobin in a dose-dependent manner, with 0.1 ng/mL bFGF inhibiting hemoglobin induction by 40% and 10 ng/mL bFGF completely abrogating hemoglobin production. bFGF was most effective at antagonizing the TGF-beta-mediated induction of hemoglobin if it and TGF-beta were added simultaneously to K562 cells, but delayed addition of bFGF to TGF-beta-treated cultures still resulted in significant inhibition of hemoglobin synthesis. The inhibitory effects of bFGF on hemoglobin production were fully reversible, showing that bFGF did not permanently alter the phenotype of K562 cells. The hemin-mediated induction of hemoglobin synthesis in K562 cells was only partially negated by bFGF. bFGF also diminished the expression of glycophorin A on the surface of K562 cells. These results indicate that bFGF might increase progenitor/stem cell numbers by antagonizing the effects of cytokines that induce differentiation, thereby increasing the pool of proliferating progenitor/stem cells.

Cell Differentiation↗
PubMed1994

Synthesis and expression of a gene coding for Erythrina trypsin inhibitor (ETI).

A gene coding for Erythrina trypsin inhibitor (ETI) was designed, based on the published N-terminal sequence of the protein, and synthesized by an oligonucleotide-directed single strand break-repair mechanism. Direct expression from the expression vector pBtac1 was unsuccessful. A construct, encoding an extended methionyl N-terminal amino acid was expressed from the vector pET12a which supplies a signal sequence for export to the periplasm. Most of the expressed protein was located in the cytoplasm but because the periplasm is an environment conducive to the formation of disulphide bridges, only periplasmic protein was extracted. Cyanogen bromide cleavage at the sole methionyl residue removed the undesired amino acid residues that remained after signal sequence peptidase processing. The resultant ETI was assayed against trypsin and tissue plasminogen activator and found to have activity similar to that of natural ETI.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of the synthetic Erythrina trypsin/tissue plasminogen activator (tPA) inhibitor encoding-gene to compare the interaction of Erythrina and soybean trypsin inhibitor with tPA.

Erythrina trypsin inhibitor (ETI) has good structural and sequence homology with soybean trypsin inhibitor (STI). However, STI does not inhibit tPA. From the three-dimensional structure of ETI it was known that the N-terminus of the molecule forms a finger-like structure stabilized by hydrogen bonds and hydrophobic interactions. In addition, the N-terminal finger region is located in close proximity to the reactive site loop and the N-terminal residue (Val) is bound up in the finger region. In STI the N-terminal region is located in close proximity to the reactive site loop and is folded into a structure similar to that in ETI. It was hypothesized that the N-terminal region is stabilized as in ETI and that the N-terminal residue of STI (Asp), because of its hydrophilic nature, is not involved in the structured N-terminal finger region of this protein. This leaves Asp1 of STI free to form an ion pair with Lys60 of trypsin, when STI and trypsin interact. When amino acid sequences of trypsin and the C-terminus of tPA are aligned for optimum homology, it is seen that there are a number of insertion sequences in tPA that are thought to be accommodated in the form of protrusions. One of these can be seen to occur in the region that lies opposite the Lys60 region of trypsin. It is suggested in this work that the N-terminal Asp of STI and this protrusion of tPA sterically prevent the two proteins from approaching close enough for binding and inhibition to occur. A modified form of ETI was produced with an Asp residue N-terminal to Val to simulate the N-terminal region of STI. The active sites were titrated against trypsin and assayed against tPA. The results showed that the modified form of ETI had activity towards tPA similar to that of STI. This evidence indicates strongly that the N-terminal Asp of STI prevents its binding to and inhibiting tPA.

Amino Acid Sequence↗
PubMed1993

Processing of HLA-class II invariant chain and expression of the p35 form is different in malignant and transformed cells.

A monoclonal antibody VCD-1, directed against the N-terminal intracellular part of the invariant chain (li) was used to show, by immunoprecipitation and Western blotting, the unprocessed and processed forms of li in chronic lymphocytic leukemia (CLL) cells, in Epstein-Barr virus-transformed normal lymphocytes (EBVL), and in cells of the Raji Burkitt's lymphoma cell line. Terminal glycosylation and sulphation of li in the Golgi apparatus was shown in Raji cells and not in EBVL. CLL lymphocytes contain a higher concentration of p35 li than do EBVL or Raji cells.

Aggrecans↗
PubMed1993

Bibrotoxin, a novel member of the endothelin/sarafotoxin peptide family, from the venom of the burrowing asp Atractaspis bibroni.

A new member of the endothelin/sarafotoxin family of vasoconstrictor peptides, bibrotoxin (BTX), was isolated from the venom of the burrowing asp Atractaspis bibroni by reversed-phase FPLC. The amino acid sequence of BTX differs from SRTX-b in the substitution Ala4 instead of Lys4, which suggests that it represents the peptide isoform of Atractaspis bibroni corresponding to SRTX-b. BTX competed for [125I]ET-1 binding to human ETB-type receptor with a Ki of 3.2 x 10(-9) M compared to 4.2 x 10(-9) M for SRTX-b. In rat thorax aorta BTX induced vasoconstrictions with a threshold concentration of 3 x 10(-8) M compared to 1 x 10(-9) for ET-1.

Amino Acid Sequence↗
PubMed1993

Absence of functional inhibition of cloned human beta 2-adrenergic receptors by autoantibodies in asthmatic subjects.

Using guinea-pig lung membranes and cloned human beta 2-receptor adrenergic receptors the effects of whole serum, plasma, purified immunoglobulins and cellular activation products on beta 2-adrenergic receptor ligand binding and function were investigated. Sera from 24 non-asthmatic subjects and 115 asthmatics in different clinical categories were studied. There were no significant differences between antagonist ([125I] cyanopindolol) inhibition mediated by serum, plasma or by purified IgG when the asthmatics were compared with non-asthmatics. There was also no inhibition of 10(-6) M isoproterenol stimulated cAMP release from L cells expressing human beta 2-adrenergic receptors, by plasma, DEAE purified IgG fractions from asthmatics and non-asthmatics, or by products of activated platelets or lymphocytes. Since we have no evidence that immunoglobulins from asthmatic subjects exert functional inhibition of human beta 2-adrenergic receptors we conclude that autoantibodies to the beta 2-adrenergic receptors do not play an important functional role in the pathophysiology of asthma.

Adrenergic beta-Agonists↗
PubMed1993

Urinary tissue plasminogen activator in renal disease.

Whereas plasminogen activator of the tissue-type (t-PA) is present in extracts of kidney parenchyma, only small amounts of the enzyme can be detected in normal urine where the major plasminogen activator is of the urokinase-type (u-PA). These observations suggest the existence of physiological or anatomical barriers that effectively confine t-PA to renal tissue and exclude it from the urine. We examined the notion that disease might breach these barriers and so lead to the appearance of abnormal amounts of t-PA in the urine. Under the conditions of the simple fibrinolytic assay that we have developed, urine samples from 30 normal subjects did not contain detectable amounts of t-PA whereas we were able to demonstrate t-PA in samples from 43 of 65 patients with various forms of renal disease. When positive, therefore, tests for the presence of t-PA in human urine provide evidence for renal disease that may not otherwise be apparent.

Adolescent↗
PubMed1992

Fibroblast-dependent tumorigenicity of melanoma xenografts in athymic mice.

Two human melanoma cell lines, UCT-Mel 2 and UCT-Mel 3, were invariably tumorigenic in nude mice when inoculated s.c. in doses of 10(6) cells or higher; 10(5) cells or less did not give rise to tumours. In this report we show that otherwise sub-tumorigenic inocula developed into vigorously growing tumour xenografts when co-inoculated with normal fibroblasts. Fibroblasts derived from adult, neonatal or embryonic tissues all functioned as complementing cells, as did cells of human or murine origin. There was, however, a requirement for complementing cell viability, since ethanol-killed fibroblasts were inefficacious. The fibroblast effect was dose-dependent and was not observed if injections of fibroblasts and melanoma cells were separated anatomically or temporally. We have shown, by titrating admixtures of melanoma cells and fibroblasts, that fibroblasts are, in quantitative terms, more efficacious than melanoma cells as complementing cells. The system we describe provides a useful model for the study of stromal-cell regulation of tumour growth.

Animals↗
PubMed1992

The desmoplastic response: induction of collagen synthesis by melanoma cells in vitro.

When cells from the human malignant melanoma cell line, UCT-Mel 7, were injected into athymic nude mice, tumours developed that were intensely infiltrated with fibrous tissue. In an attempt to reproduce this desmoplastic response in vitro, we co-cultured fibroblasts with UCT-Mel-7 cells, and observed a 2-fold increase in the rate of fibroblast collagen synthesis. This was associated with an increase in the amount of collagen mRNA present in co-cultured fibroblasts. The stimulation was both dose- and time-dependent, with maximal stimulation at a melanoma cell:fibroblast ratio of 1:1. Analysis of the kinetics of proline incorporation into collagen showed that co-culture affected the maximal rate of proline incorporation; no effect was observed on the concentration of proline required for 50% maximal collagen synthesis. The induction of fibroblast collagen synthesis showed an absolute requirement for close proximity between the fibroblasts and the melanoma cells. No soluble fibrogenic factor released by melanoma was detected.

Animals↗
PubMed1992

Unusual growth characteristics of human melanoma xenografts in the nude mouse: a model for desmoplasia, dormancy and progression.

When human melanoma cells are injected into nude mice they usually give rise to tumours that grow progressively and do not elicit a prominent host response. We have recently developed a melanoma cell line, UCT-Mel 7, that did not show these characteristics. In the first place UCT-Mel 7 showed a consistently unusual, phasic growth pattern. After a short initial period of limited growth (phase 1), the tumour ceased growing and remained static for 2-3 months (phase 2). The tumour then regressed (phase 3) to enter a second period of quiescence (phase 4) which was eventually broken by the emergence of a rapidly growing lethal tumour (phase 5). Of particular interest was the fact that the rate at which the tumours grew correlated closely with their collagen content. During the prolonged, phase 2 plateau, the tumours were intensely desmoplastic; rapidly growing phase 5 tumours, that had escaped from dormancy, contained very little collagen and virtually no reticulin. This cell line helps to fill an important need for an experimental system for the study of desmoplasia, dormancy and progression.

Animals↗
PubMed1991

The Erythrina protease inhibitor: interactions with tissue plasminogen activator.

The Kunitz-type trypsin and tissue plasminogen activator (t-PA)-inhibitor from Erythrina caffra seeds was cleaved by trypsin at low pH to yield a disulphide linked two-chain molecule with reduced hydrophobicity. This change was used to separate cleaved from native inhibitor by phenyl-Sepharose chromatography. The inhibitor was not cleaved by t-PA. Trypsin, but not t-PA, catalysed resynthesis of the cleaved bond. Although the cleaved protein retained inhibitory activity for both trypsin and t-PA, 6-10 times higher concentrations were required for equivalent inhibition. Removal of the active site arginine (Arg63) from the cleaved inhibitor by digestion with carboxypeptidase B resulted in a further loss of inhibitory activity towards both proteases. The activity of the inhibitor could also be decreased by modification of one susceptible arginine residue with peptidyl arginine deiminase. These results suggest that the trypsin-reactive site of the Erythrina inhibitor is also involved in the interaction between the inhibitor and t-PA.

Amino Acid Sequence↗
PubMed1991

Clustering of fungal allergen-specific IgE antibody responses in allergic subjects.

Statistical analysis of specific IgE immune responses to a panel of nine allergens from 2,094 patients demonstrated significant clustering between Aspergillus fumigatus, Alternaria alternata, and Cladosporium herbarum, not explained by crossreactive elements. This may represent a linked, high responder status to these fungal antigens in the allergic population.

Adult↗
PubMed1988

Metastasis of a human melanoma cell line in the nude mouse.

A human melanoma cell line has been established which when inoculated subcutaneously into nude mice, is consistently metastatic. In order to document blood-borne spread, it was necessary to excise the primary tumour so prolonging the life of the animal and allowing metastases to become apparent. Macroscopically detectable metastatic spread at autopsy was reliably indicated by weight loss of the animals. Metastases were widespread and involved the lungs, abdominal cavity and organs and the gonads. The size of the primary tumour at the time of its removal, and not the period of s.c. growth, determined the incidence of metastatic disease. Removal of tumours weighing less than 0.6 g prevented metastasis, whereas all of the animals showed widely disseminated disease if the tumour was allowed to attain a size of 1.6 g before excision.

Animals↗
PubMed1987

A monoclonal antibody to an antigen present in cells from a patient with Hodgkin's disease.

A monoclonal antibody-secreting hybridoma cell line, VCD-1, was derived from the fusion of murine myeloma cells with splenocytes from a BALB/c mouse that had been immunised with chronic B-lymphocytic leukaemia cells. The cells came from a patient who had developed the leukaemia approximately 10 years after a course of radiotherapy for nodular sclerosing Hodgkin's disease. The antibody bound to a 30,000-dalton protein that was present in normal and malignant B cells, in monocytes, neutrophils, and interdigitating reticulum cells, and in malignant cells present in Hodgkin's disease lymph nodes. The reactive epitope was not accessible to antibody in viable intact cells; binding to peripheral blood cells could only be seen if the cells were fixed. The antibody recognises a determinant that probably resides on the alpha-chain of HLA class II molecules.

Adult↗
PubMed1987

The primary structure of the inhibitor of tissue plasminogen activator found in the seeds of Erythrina caffra.

Trypsin and tissue plasminogen activator inhibitor DE-3 from Erythrina caffra contains 172 amino acids, including 4 half-cystine residues, and resembles the Kunitz-type inhibitors. Limited hydrolysis of DE-3 with trypsin at pH 3.2 produced two fragments, F1 and F2, containing 63 and 109 amino acids, respectively. Amino-terminal sequence studies showed that F1 was the N-terminal and that F2 was the C-terminal fragment. The complete amino acid sequence of the fragments were then determined on peptides produced by enzymatic digestion with trypsin. The sequence of trypsin and tissue plasminogen activator inhibitor DE-3 from E. caffra seeds shows a high degree of homology to that of trypsin and tissue plasminogen activator inhibitor DE-3 from E. latissima seeds and revealed only four amino acids which were replaced.

Amino Acid Sequence↗
PubMed1987

The reactive sites of proteinase inhibitors from Erythrina seeds.

Although the Kunitz-type proteinase inhibitors from the seeds of various Erythrina species have similar molecular weights (approximately 20,000), and share many other chemical characteristics, they could nevertheless be divided into three groups on the basis of their relative abilities to inhibit chymotrypsin, trypsin and tissue plasminogen activator. Group a inhibitors were relatively specific for chymotrypsin; they were poor inhibitors of trypsin and had no apparent effect upon tissue plasminogen activator. Group b proteins inhibited trypsin strongly and chymotrypsin slightly less effectively. They had no effect upon t-PA. Group c inhibitors inhibited trypsin, chymotrypsin and t-PA. Analysis of the amino acid composition of the three groups of inhibitors revealed major differences in alanine content. Minor differences in the content of most other amino acids were also noticed. Group b and group c inhibitors had, in most cases, the same reactive sites (Arg-Ser). The sequences neighbouring the reactive sites showed a significant degree of homology. Chemical modification of arginine in proteinase inhibitors from the seeds of E. latissima and soybeans using 1-2-cyclohexanedione confirmed the presence or absence of arginine in the reactive sites.

Amino Acid Sequence↗