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Biomedical subjects

K D Brown

Publications and source records attributed to K D Brown.

At least 55 records · Page 3Linked to original sources
PubMed1994

Activation of AT1 angiotensin receptors induces DNA synthesis in a rat intestinal epithelial (RIE-1) cell line.

Proliferation of the rat intestinal epithelial cell-line, RIE-1, has previously been shown to be stimulated by certain polypeptide growth factors acting via receptors that possess intrinsic tyrosine kinase activity. In this study, we show that the octapeptide hormone angiotensin II (AII), apparently acting through the AT1 G-protein-coupled receptor, is also a mitogen for RIE-1 cells. Maximal stimulation of DNA synthesis and cellular proliferation occurred at an AII concentration of 10-100 nM, with half-maximal stimulation at 1 nM. The mitogenic response to AII was completely inhibited by the AT1 angiotensin-receptor antagonist, DuP753, but not by the AT2-receptor antagonist, PD123319. The early signalling responses activated by AII in RIE-1 cells include increased production of inositol phosphates, a transient increase in the intracellular concentration of free calcium, an activation of protein kinase C, and a rapid change in the pattern of cellular protein-tyrosine phosphorylation. These results implicate an activation of the inositol lipid signalling pathway via the AT1 receptor subtype in the AII-stimulated mitogenic response of this normal epithelial cell line.

Angiotensin II↗
PubMed1994

Expression of c-kit and kit ligand at the human maternofetal interface.

Kit ligand, or stem cell factor, is a recently identified growth factor, which binds to and activates the c-kit proto-oncogene, and which has been shown to act synergistically with other haematopoietic growth factors in the bone marrow. We have previously shown that several isoforms of kit ligand, which arise due to alternative splicing, are expressed in human placenta. In order to elucidate the role of c-kit and its ligand during human placental development we have investigated the expression of c-kit and kit ligand in human first trimester and term placenta as well as in pregnant and non-pregnant endometrium, by immunocytochemistry and flow cytometric analysis. In non-pregnant endometrium no expression of kit ligand was seen. By contrast, in first trimester decidua, kit ligand was strongly expressed by the arterial media of maternal blood vessels. Kit ligand was also expressed throughout pregnancy by invasive fetal extravillous trophoblast, and by fetal fibroblasts within the placental villi. c-kit was found to be expressed on Hofbauer cells within the chorionic villi, and by decidual macrophages at all stages in pregnancy. c-kit was also detected on the small CD56dim subset of uterine large granular lymphocytes which form the major leukocyte population in human first trimester decidua. Our results suggest that kit ligand may be involved in the regulation of fetal macrophages, and in particular in signalling between invading extravillous trophoblast which expresses kit ligand, and maternal leukocytes bearing the c-kit receptor.

Amino Acid Sequence↗
PubMed1994

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis.

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.

Cell Cycle↗
PubMed1994

Cloning and characterization of ERF-1, a human member of the Tis11 family of early-response genes.

Members of the Tis11 family of early-response genes are characterized by a high degree of sequence similarity around a putative zinc finger motif. They are induced by a variety of cell agonists and polypeptide mitogens, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF). We describe the cloning and sequencing of a human member of this gene family, EGF-response factor 1 (ERF-1), the homolog of the mouse Tis11b/rat cMG1 genes. The human and rodent genes are similar, with 5' UTR, coding sequence, and 3' UTR highly conserved. The promoter/enhancer region and intron sequences contain multiple putative transcription factor binding motifs characteristic of early-response genes. Amino acid sequence comparison of the seven members of the Tis11 family cloned so far identifies a repeated consensus motif of (x+)YKTELC(x+)x5GxCxYGx(x+)CxFxH involving the potential zinc finger. Toward the carboxyterminal end is a region with a high percentage of prolines (15/73) and, partially overlapping, a serine-rich domain (20/54). These may be important as trans-activation and phosphorylation sites. The 3' untranslated region is unusually long, extending over 1,860 bp. The sequence immediately downstream from the translational stop codon has extensive secondary structure potential. The 3' UTR is 60% AT rich, but contains two GC rich (> 70%) regions. In addition there are multiple reiterations of a destabilization sequence, as well as a single UUAUUUAU motif characteristic of mRNAs specifying proteins involved in the inflammatory response. The mRNA contains a consensus polyadenylation signal.

Amino Acid Sequence↗
PubMed1994

Expression of the genes for the epidermal growth factor receptor and its ligands in porcine oviduct and endometrium.

We have used the reverse transcription-polymerase chain reaction (RT-PCR) to determine whether transcripts for the epidermal growth factor (EGF) receptor and its four known ligands--EGF, transforming growth factor alpha (TGF alpha), amphiregulin (Ar), and heparin-binding EGF (HB-EGF)--are expressed in porcine oviduct and endometrium. We were able to detect mRNA for the EGF receptor, EGF, TGF alpha, and Ar in both the oviduct and endometrium, whereas HB-EGF mRNA was not detectable in either tissue. Through use of an antiserum raised against recombinant pig EGF, expression of EGF was found to be localized to the columnar epithelial cells of the oviduct and to the glandular epithelial cells of the endometrium. The possible physiological roles of the EGF family in the reproductive tract are discussed.

Amphiregulin↗
PubMed1994

Elderly patients: still clean and sitting quietly.

This study investigates the activities and interactions of elderly patients in an acute medical geriatric unit and a psychiatric unit. The Clifton Assessment Procedures for the Elderly were used to measure cognitive and behavioural functioning, and the 24 subjects studied were divided into three groups: lucid, confused, and demented. Information about subjects' activities and interactions with nursing staff was gained from time sampling by non-participant direct observation. The results reveal very low levels of staff-patient interaction outside of expected routines of patient care. At no time during the observation periods did staff engage patients in social activities or prolonged informal conversations. Yet 306 nurses, of various grades, completed a questionnaire in which they ranked talking to patients as 'enjoyable', 'important', 'rewarding', and an 'objective' for themselves and the unit.

Activities of Daily Living↗
PubMed1994

Directed expression of simian virus 40 T-antigen in transgenic mice using the epidermal growth factor gene promoter.

A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5.5 kb fragment of the 5'-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5.5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.

Androgens↗
PubMed1994

Cerebral ischemia and infarction.

Cerebral infarction is most commonly related to atherosclerotic disease in the carotid and vertebrobasilar circulations. TIAs are manifestations of this same disease process and may occur before a complete infarction. The transformation of pale to hemorrhagic infarction may result from reperfusion of an infarct or from migration of an embolus. CT is often the primary imaging study to exclude frank hemorrhage. The use of MR is increasing, in both the acute and the subacute phases of stroke. The combination of MR imaging and MR angiography is gradually replacing CT, particularly in patients with TIAs. In addition, the depiction of minute alterations in contrast and subtle mass effect is visualized with MR more often than with CT. The precise localization of brain stem and posterior fossa infarctions is improved by the ability of MR to view complex structures in two or more orthogonal planes. Vascular structures are readily identified both on the spin echo images and with MR angiography. Slow or obstructed flow in the venous channels may be recognized, which makes MR the ideal examination for the evaluation of cerebral ischemia and infarction.

Brain Ischemia↗
PubMed1993

Molecular cloning and tissue distribution of pig transforming growth factor alpha.

Transforming growth factor alpha (TGF alpha) was originally identified as a product of tumour tissues and transformed cells in culture. Although it is now clear that expression of this factor is not restricted to neoplastic cells, there remains relatively little information about the sites of expression of TGF alpha in normal tissues. Therefore, an amplified DNA fragment encoding the pig TGF alpha precursor was cloned by reverse transcription-PCR (RT-PCR) using RNA isolated from normal skin tissue as the template. Nucleotide sequence analysis predicts a 160-residue transmembrane polypeptide that differs from the rat, mouse and human TGF alpha precursors at 14, 15 and six sites respectively. The distribution of TGF alpha mRNA in a wide variety of pig tissues was analysed by RT-PCR, using oligonucleotide primers based on the pig TGF alpha cDNA sequence. TGF alpha transcripts were detected in RNA isolated from 17 of the 22 tissues analysed, including four previously unreported sites. Using an antibody raised against a synthetic TGF alpha peptide, we have immunolocalized TGF alpha protein to cells within the red pulp of the spleen and to the distal convoluted tubules of the kidney.

Amino Acid Sequence↗
PubMed1993

Actin-binding protein is a component of bovine erythrocytes.

Actin-binding protein (ABP) is a well-characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical.

Animals↗
PubMed1993

Expression of the cytoskeletal-associated protein filamin in adult rat organs.

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.

Animals↗
PubMed1993

Activation of an imprinted Igf 2 gene in mouse somatic cell cultures.

The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture. MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth. Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression. Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine.

Animals↗
PubMed1993

Expression of the genes for the epidermal growth factor receptor and its ligands in porcine corpora lutea.

Using reverse transcription-polymerase chain reaction (RT-PCR), transcripts for EGF receptor and for EGF, transforming growth factor-alpha (TGF alpha), amphiregulin (AR) and heparin binding-EGF (HB-EGF) were detected in porcine corpora lutea at day 4, 10 and 20 of the estrous cycle. No cycle-dependent changes were apparent in the expression of the receptor, AR or TGF alpha. In contrast, HB-EGF mRNA levels appeared highest on day 4 during luteal growth and development, whereas EGF mRNA levels appeared highest on day 20 when the corpora lutea undergo luteolysis. These results suggest that luteal function may be subject to regulation by members of the EGF family acting in an autocrine, paracrine or intracrine manner.

Animals↗
PubMed1993

Expression of mRNA for vascular endothelial growth factor in human placenta.

Implantation and growth of the placenta requires extensive angiogenesis to establish the vascular structures involved in exchange. Failure to establish adequate blood supply to the fetus may have serious clinical consequences such as intrauterine growth retardation. Vascular endothelial cell growth factor (VEGF) is a recently identified growth factor with significant angiogenic properties. We have demonstrated the presence of four species of mRNA encoding VEGF in both first trimester and term placenta. In situ hybridization was used to localize the sites of expression of VEGF mRNA in these tissues. VEGF expression was seen in villous trophoblast in the first trimester and in extravillous trophoblast at term, and in both fetal macrophages within the villi and maternal macrophages in the decidua. Glandular epithelium in maternal decidua also expressed VEGF mRNA. The strongest site of expression was in maternal macrophages adjacent to Nitabuch's stria, a zone of necrosis at the site of implantation. This complex pattern of expression suggests that VEGF is involved in angiogenesis on both maternal and fetal sides of the placenta and that macrophages are the primary source of VEGF. However, VEGF may also play a role in term placenta, when extensive angiogenesis has diminished, possibly regulating vascular permeability.

Base Sequence↗
PubMed1992

Tissue distribution of mRNA for heparin-binding epidermal growth factor.

Heparin-binding epidermal growth factor (HB-EGF) is a recently identified member of the EGF family. Mature HB-EGF is processed from a larger transmembrane precursor which can itself act as a cell-surface receptor for the internalization of diphtheria toxin into eukaryotic cells. However, to date there is no information available on the distribution of HB-EGF in mammalian tissues. We have therefore used reverse-transcription PCR to analyse the expression of HB-EGF mRNA in a wide range of tissues. HB-EGF transcripts were detected in RNA isolated from 15 of the 22 tissues obtained from adult pigs, which is consistent with the ability of diphtheria toxin to affect many body tissues.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence and tissue distribution of mouse transforming growth factor-alpha.

Transforming growth factor-alpha (TGF alpha) is secreted into the medium of the Moloney murine sarcoma virus-transformed 3T3 cell line, 3B11-1C. Using reverse transcription-polymerase chain reaction (RT-PCR), we have amplified, cloned and sequenced a cDNA fragment from this cell line which encodes the full protein-coding region of the mouse TGF alpha precursor. The deduced amino acid sequence (159 residues) differs from that of human TGF alpha at 13 sites and from that of rat at 3 sites. In the mouse, TGF alpha transcripts were detected in a variety of normal adult tissues including two previously unreported sites: the preputial glands and the bladder.

Amino Acid Sequence↗