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Biomedical subjects

D J Riley

Publications and source records attributed to D J Riley.

At least 37 records · Page 2Linked to original sources
PubMed1997

Polymer of proline analogue with sustained antifibrotic activity in lung fibrosis.

Inhibitors of collagen such as cis-4-hydroxy-L-proline (cHyp) may ameliorate bleomycin (bleo)-induced pulmonary fibrosis. An alternating polymer of poly(ethylene glycol) (PEG)-lysine (PEG-Lys) with cHyp attached as a pendant side chain was prepared for intratracheal delivery with bioinactive trans-Hyp (tHyp) polymer as control. To test whether the cHyp polymer has prolonged lung retention and sustained antifibrotic activity, we first instilled 3H- and 14C-labeled cHyp polymer in normal rats. Lung retention was 86 +/- 9% at 6 h and 29 +/- 3% at 7 d (n = 5). Next, rats were instilled intratracheally with either saline (sal) or 1.2 U bleo, and the following treatment groups were studied: Bleo/sal; Bleo/cHyp polymer; Bleo/tHyp polymer; and Bleo/PEG-Lys + cHyp. The dose of the test agents was 150 mg/kg polymer containing 8.5 mg/kg cHyp or tHyp instilled intratracheally at 7 and 14 d after bleo. At 21 d, hydroxyproline content (mg/lung) was: Control, 1.8 +/- 0.1; Bleo/sal 4.0 +/- 0.1*; Bleo/cHyp polymer, 2.8 +/- 0.3*+; Bleo/tHyp polymer, 4.4 +/- 0.2*; and Bleo/PEG-Lys + cHyp, 4.0 +/- 0.1* (*p < 0.05 versus Control; +p < 0.05 versus Bleo/sal; n = 5/group). The cHyp polymer also reduced lung total protein content, but the decrease was not significant. The dose required to produce 50% inhibition of lung collagen was approximately 700-fold less than monomeric cHyp. Thus, the cHyp polymer is a potent, long-acting antifibrotic agent which may be useful in treating lung fibrosis.

Animals↗
PubMed1996

Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs.

To define a mechanism by which retinoblastoma protein (Rb) functions in cellular differentiation, we studied primary fibroblasts from the lung buds of wild-type (RB+/+) and null-mutant (RB-/-) mouse embryos. In culture, the RB+/+ fibroblasts differentiated into fat-storing cells, either spontaneously or in response to hormonal induction; otherwise syngenic RB-/- fibroblasts cultured in identical conditions did not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enabled RB-/- fibroblasts to differentiate into adipocytes. Rb appears in murine fibroblasts to activate CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors crucial for adipocyte differentiation. Physical interaction between Rb and C/EBPs was demonstrated by reciprocal coimmunoprecipitation, but occurred only in differentiating cells. Wild-type Rb also enhanced the binding of C/EBP to cognate DNA sequences in vitro and the transactivation of a C/EBPbeta-responsive promoter in cells. Taken together, these observations establish a direct and positive role for Rb in terminal differentiation. Such a role contrasts with the function of Rb in arresting cell cycle progression in G1 by negative regulation of other transcription factors like E2F-1.

Adipocytes↗
PubMed1996

Retinoblastoma protein directly interacts with and activates the transcription factor NF-IL6.

The biological function of the retinoblastoma protein (RB) in the cell division cycle has been extensively documented, but its apparent role in differentiation remains largely unexplored. To investigate how RB is involved in differentiation, the U937 large-cell lymphoma line was induced to differentiate along a monocyte/macrophage lineage. During differentiation RB was found to interact directly through its simian virus 40 large tumor antigen (T antigen)-binding domain with NF-IL6, a member of the CAAT/enhancer-binding protein (C/EBP) family of transcription factors. NF-IL6 utilizes two distinct regions to bind to the hypophosphorylated form of RB in vitro and in cells. Wild-type but not mutant RB enhanced both binding activity of NF-IL6 to its cognate DNA sequences in vitro and promoter transactivation by NF-IL6 in cells. These findings indicate a novel biochemical function of RB: it activates, by an apparent chaperone-like activity, specific transcription factors important for differentiation. This contrasts with its sequestration and inactivation of other transcription factors, such as E2F-1, which promote progression of the cell cycle. Such disparate mechanisms may help to explain the dual role of RB in cell differentiation and the cell division cycle.

Amino Acid Sequence↗
PubMed1996

Adenovirus-mediated retinoblastoma gene therapy suppresses spontaneous pituitary melanotroph tumors in Rb+/- mice.

The retinoblastoma gene (RB) is the prototypic tumor suppressor. Studies to date have demonstrated cancer suppression with tumor cells reconstituted with RB ex vivo and implanted into immunodeficient mice, as well as with germline transmission of a human RB transgene into tumor-prone Rb +/- mice. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb +/- mice were treated with a recombinant adenovirus carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, reestablished innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life spans of treated animals.

Adenoviruses, Human↗
PubMed1996

A new member of the hsp90 family of molecular chaperones interacts with the retinoblastoma protein during mitosis and after heat shock.

A gene encoding a new heat shock protein that may function as a molecular chaperone for the retinoblastoma protein (Rb) was characterized. The cDNA fragment was isolated by using the yeast two-hybrid system and Rb as bait. The open reading frame of the longest cDNA codes for a protein with substantial sequence homology to members of the hsp90 family. Antibodies prepared against fusions between glutathione S-transferase and portions of this new heat shock protein specifically recognized a 75-kDa cellular protein, hereafter designated hsp75, which is expressed ubiquitously and located in the cytoplasm. A unique LxCxE motif in hsp75, but not in other hsp90 family members, appears to be important for binding to the simian virus 40 T-antigen-binding domain of hypophosphorylated Rb, since a single mutation changing the cysteine to methionine abolishes the binding. In mammalian cells, Rb formed complexes with hsp75 under two special physiological conditions: (i) during M phase, when the envelope that separates the nuclear and cytoplasmic compartments broke down, and (ii) after heat shock, when hsp75 moved from its normal cytoplasmic location into the nucleus. In vitro, hsp75 had a biochemical activity to refold denatured Rb into its native conformation. Taken together, these results suggest that Rb may be a physiological substrate for the hsp75 chaperone molecule. The discovery of a heat shock protein that chaperones Rb identifies a mechanism, in addition to phosphorylation, by which Rb is regulated in response to progression of the cell cycle and to external stimuli.

Amino Acid Sequence↗
PubMed1995

Aberrant subcellular localization of BRCA1 in breast cancer.

The BRCA1 gene product was identified as a 220-kilodalton nuclear phosphoprotein in normal cells, including breast ductal epithelial cells, and in 18 of 20 tumor cell lines derived from tissues other than breast and ovary. In 16 of 17 breast and ovarian cancer lines and 17 of 17 samples of cells obtained from malignant effusions, however, BRCA1 localized mainly in cytoplasm. Absence of BRCA1 or aberrant subcellular location was also observed to a variable extent in histological sections of many breast cancer biopsies. These findings suggest that BRCA1 abnormalities may be involved in the pathogenesis of many breast cancers, sporadic as well as familial.

Amino Acid Sequence↗
PubMed1995

Regulatory networks of the retinoblastoma protein.

Studies of the retinoblastoma (RB) gene product suggest that it may work as a fundamental regulator to coordinate pathways of cellular growth and differentiation. One known function of retinoblastoma (Rb) protein is its ability to suppress tumorigenesis. In many different cultured tumor cells, replacement of a normal RB gene and expression of normal Rb protein results in suppression of neoplastic properties. Moreover, in humans or experimental mice, germ line mutation of the RB gene leads particularly to retinoblastomas or pituitary tumors, respectively, which demonstrates that the role of RB in tumor predisposition is specific to certain tissues. In addition to suppressing tumor formation, Rb apparently also has roles in normal development and cellular differentiation. Recent characterizations of Rb-associated proteins and proteins within the Rb family may provide some clues to exploring the complex networks in which Rb is involved.

Animals↗
PubMed1995

Elastin gene mutations in transgenic mice.

We have constructed several rat tropoelastin minigene recombinants encoding the complete sequence of rat tropoelastin, two isoforms of rat tropoelastin and a truncated tropoelastin lacking the domains encoded by exons 19-31 of the rat gene. Coding and non-coding domains in all these recombinants were placed under the transcriptional control of 3 kb of the promoter domain of the rat tropoelastin gene. These minigenes were used to prepare a total of 28 separate founder lines of transgenic mice. A species-specific reverse-transcriptase polymerase chain reaction (RT-PCR) assay was established to demonstrate the synthesis of rat and mouse tropoelastin mRNA in several tissues obtained from both neonatal and adult transgenic mice. Thermolytic digestion of insoluble elastin isolated from several neonatal mouse tissues revealed the presence of rat tropoelastin peptides in progeny from all those founder mice in which detectable levels of rat tropoelastin mRNA were noted. Phenotypic and histopathological assessment of transgenic and non-transgenic animals revealed the development of two diverse elastic tissue disorders. The progeny of two separate founder lines overexpressing the rat tropoelastin isoform lacking exon 33, developed an emphysematous phenotype in early adulthood. In contrast, transgenic mice, in which expression of the truncated rat tropoelastin minigene lacking exons 19-31 had been observed, died of a ruptured ascending aortic aneurysm. Tropoelastin gene mutations, therefore, will result in heritable disorders of elastic tissue. Moreover, different mutations in the tropoelastin gene will be responsible for very different abnormalities in elastic tissue function.

Animals↗
PubMed1995

Effect of diet on lung structure, connective tissue metabolism and gene expression.

Nutritional impairment can adversely affect the respiratory system. The structure of the lung is altered by severe calorie-protein restriction in rodents producing an emphysema-like lesion after several weeks of severe caloric restriction. Biochemical and morphological evidence suggests destruction of collagen and elastin in nutritional emphysema. Impaired lung growth may explain the biochemical changes in growing animals. Although the molecular basis of nutritional emphysema is not known, altered gene expression by nutrients affects several metabolic pathways, and examples of the effects of nutrients on gene expression are given. Nutritional emphysema in animals may be relevant to humans because malnutrition may accelerate the progression of the disease in patients with advanced emphysema.

Animals↗
PubMed1995

The potential of gene therapy for treatment of kidney diseases.

As knowledge of the genes and molecular events involved in specific kidney disorders expands, so do the potential applications for treating the disorders on fundamental levels. Progress in gene therapy for genetic diseases for which molecular events are simpler or better characterized has already been substantial. In addition, exciting first steps in targeting genes specifically to the kidney have emerged, although much work in defining kidney-specific genes and gene regulatory elements still needs to be done. To provide a framework for understanding the concepts and problems involved in gene therapy, we will discuss basic aspects of gene structure and regulation, gene delivery vectors, steps necessary to achieve tissue- and cell-specific expression of delivered genes, and some present and future applications of gene therapy in kidney diseases.

Animals↗
PubMed1995

The retinoblastoma protein as a fundamental mediator of growth and differentiation signals.

The retinoblastoma gene (RB) is the prototype of the tumor suppressor genes, which play critical roles in the genesis of cancer in humans. Mouse models created through gene knock-out and transgenic methods were established for exploring and manipulating RB in vivo. These models and several other pieces of evidence have shown that the retinoblastoma protein (Rb) plays dual roles in gating cell cycle progression and promoting cellular differentiation. The molecular mechanisms involved in these roles are becoming more obvious in some biological systems: Rb sequesters the transcription factor of E2F to regulate entry of cell cycle but enhances the activities of another class of the transcription factors, exemplified by NF-IL6, to initiate terminal cellular differentiation. Thus, the Rb protein can serve as a mediator for extracellular signals of growth or differentiation. The fundamental question of why only limited cell types are susceptible to tumor formation when Rb expression is lost, however, remains unanswered at present.

Animals↗
PubMed1994

Susceptibility to tumors induced in mice by ethylnitrosourea is independent of retinoblastoma gene dosage.

The retinoblastoma gene (RB) is a classical tumor suppressor. Several studies have shown that RB dosage is important in determining biological effects. To explore the effect of RB dosage on susceptibility to cancer, three groups of congenic C57BL/6 mice, each of which expresses a different amount of Rb protein from one, two, or three alleles, were treated at postnatal day 12 with a single 60-mg/kg body weight i.p. dose of the DNA-alkylating agent N-ethyl-N'-nitrosourea (ENU). Mice heterozygous for the RB gene developed characteristic pituitary tumors with nearly complete penetrance, whether or not they were treated with ENU. Tumors initiated earlier or progressed more rapidly, however, in ENU-treated mice. Furthermore, although mice treated with ENU had a higher incidence of several nonpituitary tumors compared with untreated controls, no significant differences in the incidence of these tumors were found between wild-type mice (mRB+/+), mice carrying only one normal RB allele and deficient in Rb protein expression (mRB+/-), and mice overexpressing Rb protein from two normal murine RB alleles and a human RB transgene (mRB+/+, hRB+/-). These studies underscore the tissue and mechanistic specificity of tumor predisposition caused by an inherited 50% reduction in RB dosage and indicate that most ENU-induced tumors occur independent of RB inactivation. Nonetheless, they suggest that certain point mutations induced by ENU may participate in the sequence of molecular steps involved in progression of tumor-prone, RB-deficient cells to the fully malignant state.

Animals↗
PubMed1994

Effect of glucocorticoids on collagen accumulation in pulmonary vascular remodeling in the rat.

Administration of corticosteroids may attenuate the development of pulmonary hypertension by inhibiting the cell proliferation and protein synthesis that occur in early pulmonary vascular remodeling. However, in vitro studies show that corticosteroids stimulate collagen synthesis in vascular smooth muscle cells, and corticosteroid administration may be deleterious in stimulating collagen deposition. To test whether corticosteroid treatment promotes vascular collagen production in vivo, we administered triamcinolone diacetate to rats exposed to 10% O2 for 3 days and measured pro alpha 1(I) collagen mRNA and the hydroxyproline/protein ratio in the main pulmonary artery. Triamcinolone treatment (12 mg/kg intraperitoneally, once daily for 3 days) reduced mean right ventricular pressure (11 +/- 1 versus 14 +/- 1 mm Hg) and protein content of pulmonary arteries (1.8 +/- 0.1 versus 2.7 +/- 0.1 mg/vessel) (both p < 0.05). However, corticosteroid treatment produced a dose-related increase in pro alpha 1(I) mRNA levels and increased the ratio of hydroxyproline/protein (47 +/- 2 versus 38 +/- 3 micrograms/mg; p < 0.05). Thus, corticosteroid administration ameliorated the increase in pulmonary hypertension in early hypoxia, but increased the proportion of collagen in the vessel wall. Corticosteroid treatment in pulmonary vascular remodeling may be deleterious in increasing the concentration of collagen in the vessel wall.

Analysis of Variance↗
PubMed1994

Excess collagen in hypertensive pulmonary arteries decreases vascular distensibility.

Increased vascular collagen content is a major feature of pulmonary vascular remodeling. The functional role of excess collagen in decreasing pulmonary vascular compliance has not been established. We determined whether there was a correlation between hydroxyproline content of rat pulmonary artery segments and elastance (EPA) of the pulmonary artery bed during development of hypoxic pulmonary hypertension (10% O2, 10 d) and normoxic recovery. EPA was measured by air-filled pressure-volume curves. After 10 d of hypoxia, hydroxyproline content increased approximately 2-fold in large segments (1,200-250 microns in diameter) but not significantly in small segments (> 250 microns). Elastance increased from 87 +/- 6 (SEM) to 145 +/- 8 mm Hg/ml (p < 0.05) within 5 d of hypoxia and returned to control value 3 wk after recovery. There was a correlation between collagen content and EPA in large segments during development of hypertension; no correlation was found during recovery from hypoxia. The ratio of hydroxyproline to total protein was unchanged in large segments after recovery from hypoxia but was increased in small segments after recovery. We conclude that increased collagen in large pulmonary arteries directly influences EPA during the development of hypoxic pulmonary hypertension.

Animals↗
PubMed1994

Liposome encapsulation improves the effect of antifibrotic agent in rat lung fibrosis.

We studied whether the therapeutic efficacy of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) in preventing bleomycin-induced pulmonary fibrosis in rats is enhanced by intratracheal delivery in liposomes. Dual-radiolabeled liposomes were used to study the distribution and stability of liposomes after intratracheal instillation. Lung retention was > 20% 1 wk after intratracheal instillation of 9 mumol phospholipid, and liposomes were intact as indicated by the ratio of the lipid and aqueous-phase markers remaining unchanged. For the fibrosis study, groups of rats were instilled with 1.2 U bleomycin (Bleo) and treated 1 and 2 wk later by single intratracheal instillation of test compounds. The control group received 0.3 ml saline (Bleo/sal). The treated groups received 9 mumol phospholipid in 0.3 ml of the following liposome preparations: empty liposomes (Bleo/lip), liposomes and 100 mg/kg of free unencapsulated cHyp (Bleo/lip/cHyp), and 100 mg/kg of liposome-encapsulated cHyp (Bleo/lip-cHyp). At 3 wk, fibrosis (mg hydroxyproline/g weight lung) by groups was as follows: control, 2.6 +/- 0.1 (SEM); Bleo/sal, 3.2 +/- 0.1, Bleo/lip, 3.2 +/- 0.1, and Bleo/lip/cHyp, 3.1 +/- 0.1, p < 0.05 compared with control; Bleo/lip-cHyp, 2.6 +/- 0.1, p < 0.05 compared with Bleo/sal, n = 3 to 6. Histologic grading of fibrosis did not show decreased fibrosis in the Bleo/lip-cHyp group, probably because of the focal nature of the fibrotic lesions. We conclude that cHyp encapsulated in liposomes prevents bleomycin-induced fibrosis by biochemical measurements. Delivery of antifibrotic agents to the lung in carrier vehicles promotes retention and may enhance their efficacy in treating bleomycin-induced pulmonary fibrosis.

Animals↗
PubMed1994

Activation of alveolar macrophages by native and synthetic collagen-like polypeptides.

Interstitial connective tissue fragments are known to be chemotactic for neutrophils and these have been implicated in mediating the migration of inflammatory cells to the lung following injury. In the present studies, we determined if degradation products of collagen also induced chemotaxis and functional activation of alveolar macrophages. For these studies, we used bovine dermal collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), glycine (Gly), and hydroxyproline (Hyp). We found that collagenase or cyanogen bromide digests of native collagen, as well as synthetic polypeptides containing Pro and Gly in the pentameric (Pro-Pro-Gly)5 form, were potent chemoattractants for rat alveolar macrophages inducing migration in the nanomolar concentration range. We also found that native and synthetic collagen peptides stimulated the release of superoxide anion and hydrogen peroxide, as well as elastase and gelatinase release from alveolar macrophages. These effects were dose and time dependent, reaching a maximum after 72 h with 0.1 to 1 microM peptides. In contrast to chemotaxis, synthetic peptides containing Hyp also stimulated reactive oxygen intermediate and elastase release from the cells. Although the pentameric and decameric forms of the synthetic peptides were equally effective in stimulating elastase release, (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 peptides were more active in inducing a respiratory burst. We also determined if alveolar macrophages were activated for cytotoxicity by collagen peptides. Treatment of the macrophages with native collagen digests or (Pro-Pro-Gly)5 was found to induce cytotoxicity of these cells towards both transformed and nontransformed rat-derived targets.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Circulating fibroblast growth factor-like autoantibodies in two patients with multiple endocrine neoplasia type 1 and prolactinoma.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. bFGF lacks a classical amino-terminal signal sequence and is not readily detectable in plasma from normal subjects. In earlier studies we showed increased bFGF-like mitogenic activity for parathyroid-derived endothelial cells and (increased) bFGF immunoreactivity (0.24-1.28 ng/mL) in plasma of subjects with multiple endocrine neoplasia type 1 (MEN-1). In the present study we examined the proliferative activity of MEN-1 and normal plasmas (applied to protein-A columns) in calf pulmonary artery endothelial cells. Protein-A-eluted activity in plasma from MEN-1 prolactinoma plasma exceeded activity from normal and MEN-1 nonprolactinoma plasma in three of eight MEN-1 subjects with untreated or recurrent prolactinoma. Protein-A-eluted active fractions from MEN-1 prolactinoma plasma had several properties of an immunoglobulin G, including affinity for antihuman immunoglobulin G (IgG) agarose, sensitivity to thiols, and (prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions) apparent mol wt corresponding to those of the heavy and light chains of IgG. The IgG fraction of MEN-1 prolactinoma plasma had far more activity in endothelial cells than did optimal concentrations of known growth factors or conditioned medium from prolactinoma cells. Endothelial cell bioactivity in protein-A-eluted fractions from MEN-1 prolactinoma plasma was neutralized 70% by rabbit antibodies to intact bFGF. These results imply novel growth stimulatory bFGF-like autoantibodies in a subset of MEN-1 patients with prolactinoma.

Adult↗