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Biomedical subjects

D Brown

Publications and source records attributed to D Brown.

At least 595 records · Page 33Linked to original sources
PubMed1993

Endosomal pathways for water channel and proton pump recycling in kidney epithelial cells.

The plasma membrane composition of virtually all eukaryotic cells is maintained and continually modified by the recycling of specific protein and lipid components. In the kidney collecting duct, urinary acidification and urinary concentration are physiologically regulated at the cellular level by the shuttling of proton pumps and water channels between intracellular vesicles and the plasma membrane of highly specialized cell types. In the intercalated cell, hydrogen ion secretion into the urine is modulated by the recycling of vesicles carrying a proton pumping ATPase to and from the plasma membrane. In the principal cell, the antidiuretic hormone, vasopressin, induces the insertion of vesicles that contain proteinaceous water channels into the apical cell membrane, thus increasing the permeability to water of the epithelial layer. In both cell types, 'coated' carrier vesicles are involved in this process, but whereas clathrin-coated vesicles are involved in the endocytotic phase of water channel recycling, the transporting vesicles in intercalated cells are coated with the cytoplasmic domains of the proton pumping ATPase. By a combination of morphological and functional techniques using FITC-dextran as an endosomal marker, we have shown that recycling endosomes from intercalated cells are acidifying vesicles but that they do not contain water channels. In contrast, principal cell vesicles that recycle water channels do not acidify their lumens in response to ATP. These non-acidic vesicles lack functionally important subunits of the vacuolar proton ATPase, including the 16 kDa proteolipid that forms the transmembrane proton pore. Because these endosomes are directly derived via clathrin-mediated endocytosis, our results indicate that endocytotic clathrin-coated vesicles are non-acidic compartments in principal cells. In contrast, recycling vesicles in intercalated cells contain large numbers of proton pumps, arranged in hexagonally packed arrays on the vesicle membrane. These pumps are inserted into the apical plasma membrane of A-type (acid-secreting) intercalated cells, and the basolateral plasma membrane of B-type (bicarbonate-secreting) cells in the collecting duct. Both apical and basolateral targeting of H(+)-ATPase-containing vesicles in these cells may be directed by microtubules, because polarized insertion of the pump into both membrane domains is disrupted by microtubule depolymerizing agents. However, the basolateral localization of other transporting proteins in intercalated cells, including the band 3-like anion exchanger and facilitated glucose transporters, is not affected by microtubule disruption.

Animals↗
PubMed1993

Abnormalities in rCBF and computed tomography in patients with Alzheimer's disease and in controls.

The pattern of abnormal distribution of the single photon emission computed tomography (SPECT) cerebral blood flow tracer 99m-technetium-hexamethylpropyleneamine oxime (99Tcm-HMPAO) was investigated in 14 patients with clinically diagnosed Alzheimer's disease (AD) who subsequently had post-mortem confirmation of the disease and also in 14 elderly control subjects. These abnormalities were compared with computed tomography (CT) scans to investigate the degree to which the focal SPECT deficits were due to atrophy. The results show that SPECT imaging with 99Tcm-HMPAO and CT scanning both have a higher incidence of abnormality in AD patients than in controls and that the difference between patients and controls is greater with SPECT than with CT. Frontal SPECT and CT abnormalities in moderate/severe Alzheimer's disease occur as frequently as temporal/occipital abnormalities but the latter are rare in control subjects. Around 50% of the SPECT deficits occur in CT normal brain regions, showing that atrophy is not the sole cause of SPECT deficits.

Aged↗
PubMed1993

Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors.

We examined the metalloproteinase activity from normal and keratoconus corneal extracts. No differences were detected in the total amount of the metalloproteinase or its physical form of activation. However, there was a significant elevation in enzymatic activity in the keratoconus extracts after chemical modification of inhibitory elements. This suggests either a difference in the enzymatic capabilities of keratoconus corneas or, as suggested previously, a decrease in the amount of TIMP (tissue inhibitor of metalloproteinase) present in the tissue.

Cells, Cultured↗
PubMed1993

Characterization of a human corneal metalloproteinase inhibitor (TIMP-1).

The gradual corneal thinning seen in keratoconus may be due to altered degradation of the corneal extracellular matrix. Studies have shown that human keratocytes produce matrix metalloproteinase-2 (MMP-2) and two proteins (28 kDa and 21 kDa) that are capable of inhibiting the activity of MMP-2. In the present study, the 28 kDa inhibitor from keratoconus keratocyte cultures has been characterized as it may be important to the elevated MMP-2 activity seen in these cultures. Biochemical analyses indicated that this keratoconus corneal inhibitor was similar to TIMP-1 from other sources. Oligonucleotides to the reported sequence of human tumor cell TIMP-1 were used for reverse-transcriptase PCR to generate a 700 bp clone of the 28 kDa inhibitor from keratoconus keratocyte cytoplasmic RNA. Sequence analysis verified that the clone was nearly identical to the reported human TIMP-1 with a single base substitution that did not affect the predicted amino acid sequence. In addition, protein translated from the clone corresponded to the expected size. This data suggests that the elevated levels of gelatinolytic activity in these keratoconus keratocyte cultures is not due to a primary alteration of the TIMP-1 molecule. Protein expression studies of the TIMP-1 clone are currently underway.

Amino Acid Sequence↗
PubMed1993

Quantification of apoptosis and necrosis by flow cytometry.

Apoptosis and necrosis are two important mechanisms of cell death. Several methods have recently been described for quantifying apoptotic cells by flow cytometry. We report a novel method for the quantification and separation of viable normal and apoptotic cells. We have applied this method both to immature rat thymocytes treated with a variety of agents and to a murine haemopoetic cell line after withdrawal of a growth factor. The cells were incubated with two dyes which give fluorescent complexes when bound to DNA, the bis-benzimidazole, Hoechst 33342, and propidium iodide. Three populations were identified and characterized. On excitation with UV radiation, dead cells fluoresced red due to the uptake of propidium iodide whereas apoptotic cells fluoresced bright blue; normal cells showed low blue, low red fluorescence. In this paper, we demonstrate how this method may be used to help to distinguish between cell death by apoptosis and necrosis.

Animals↗
PubMed1993

A second mutation associated with apparent beta-hexosaminidase A pseudodeficiency: identification and frequency estimation.

Deficient activity of beta-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C739-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second "disease-causing" allele, results in Hex A pseudodeficiency. We examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C739-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G805-to-A (Gly269Ser) mutation associated with adult-onset GM2 gangliosidosis was found on one chromosome. A new mutation, C745-to-T (Arg249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of GM2 gangliosidosis, any phenotype must be very mild. This new mutation and the benign C739-to-T mutation together account for approximately 38% of non-Jewish enzyme-defined carriers. Because carriers of the C739-to-T and C745-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided.

Adult↗
PubMed1993

Cytoskeletal changes associated with cell motility.

This chapter reviews various aspects of changes in cytoskeletal organization which occur upon activating a highly motile cell phenotype. The first of these relates to the rapid formation of F-actin-rich ruffles on the apical cell surfaces following the addition of one of several motility factors. In a number of aspects these ruffles resemble leading edge lamellipodia. The ruffles form upon ligand binding and, at least for one cytokine, the receptors become associated with the ruffles. For several cytokines the ruffles are circular and in all cases they are associated with much increased pinocytosis. The possible significance is considered of these very early markers of a motile cell phenotype. A second topic covered is the role of microtubules (MTs) in maintaining cell polarity in some cell types but not others. The micro-injection of biotin-tubulin into cells has provided a valuable marker of MT turnover. Using this method it has been found that the microtubule network in secondary chick heart fibroblasts (2 degrees CHFs), where MTs are required to maintain a polarized motility, does not turn over significantly more slowly than in 1 degree CHFs which do not require an intact MT network for locomotion. In both cases the MT network turns over very quickly and no sub-population of longer-lived MTs has been found. In contrast, in motile epithelial (PtK2) cells, a sub-population of longer-lived microtubules has been identified and these appear to maintain the long cell processes.

Animals↗
PubMed1993

Location of gp330/alpha 2-m receptor-associated protein (alpha 2-MRAP) and its binding sites in kidney: distribution of endogenous alpha 2-MRAP is modified by tissue processing.

The alpha 2-macroglobulin receptor-associated protein (alpha 2-MRAP) is a 39 to 44 kDa protein that copurifies with the alpha 2-macroglobulin receptor (alpha 2-MR/LRP) and also with gp330, a highly glycosylated protein located within kidney proximal tubules and glomerular podocytes. Both gp330 and the alpha 2-macroglobulin receptor are members of the low density lipoprotein receptor family but the physiological ligands for gp330 are unknown. In order to understand potential functions of the alpha 2-MRAP, specific anti-alpha 2-MRAP antibodies were used for immunocytochemical studies on paraformaldehyde lysine periodate (PLP)-fixed rat kidneys and on snap-frozen/acetone-fixed tissue. Conflicting results were obtained. After PLP fixation, alpha 2-MRAP was detected almost exclusively in rough endoplasmic reticulum (RER) cisternae; cell surface staining was virtually absent. In snap-frozen tissue, intense staining of the proximal tubule brush border was found, with little or no cytoplasmic staining. A series of experiments showed that during incubation of snap-frozen tissues, endogenous alpha 2-MRAP is released in soluble form from its intracellular location (i.e., the RER) and binds to gp330 on the brush border of proximal tubules. The location of binding sites for alpha 2-MRAP in rat kidney was also examined, using an alpha 2-MRAP-IgG fusion protein. In both snap-frozen and PLP-fixed tissues, this probe bound exclusively to brush borders, and not to intracellular sites. Our results demonstrate: a) that in renal proximal tubule cells, alpha 2-MRAP is located predominantly in the RER, b) that alpha 2-MRAP-binding sites are present on gp330, which is on the proximal tubule brush border, and c) that the apparent brush border localization of alpha 2-MRAP detected in snap-frozen sections is due to an artifactual redistribution of endogenous alpha 2-MRAP that occurs during tissue processing.

Animals↗
PubMed1993

Localization of the CHIP28 water channel in reabsorptive segments of the rat male reproductive tract.

The water channel protein CHIP28 is responsible for the high constitutive plasma membrane permeability to water of erythrocytes, renal proximal tubule, and thin descending limb of Henle. The male reproductive tract is embryologically related to kidney and some segments, particularly the efferent ducts, exhibit a high rate of solute flux-dependent reabsorption of luminal fluid. To determine whether this could occur through water channels in the plasma membrane of reproductive tract epithelial cells, we used anti-CHIP28 antibodies to localize this protein by Western blotting and immunocytochemistry. Western blotting of proteins from efferent duct homogenate indicated the presence of CHIP28 in the efferent duct cells. By indirect immunofluorescence and protein A-gold immunolabeling, CHIP28 was localized to the brush-border and basolateral membranes of nonciliated cells. Ciliated cells in the same epithelium showed no plasma membrane staining for CHIP28. In accord with immunocytochemical findings, freeze-fracture of nonciliated efferent duct cells revealed a plasma membrane organization resembling that of renal proximal tubule cells that are rich in CHIP28. The anti-CHIP28 antibodies also stained plasma membranes of epithelial cells in the ampulla of the vas deferens, seminal vesicles, and prostate, but not the cells in seminiferous tubules, epididymis, and proximal parts of the vas deferens. Therefore, CHIP28 may be a principal mediator of the transmembrane water transport in absorptive epithelial cells of efferent ducts, as well as in epithelial of several other segments of the male reproductive tract that show both secretory and reabsorptive functions.

Absorption↗
PubMed1993

Possible models describing enterocyte replacement in mouse Peyer's patch follicle-associated epithelial tissue.

Epithelial cells emerging from intestinal crypts surrounding Peyer's patch lymphoid follicles can be shown in chimaeric mice to adopt a wedge-like formation during migration to a central zone of cell extrusion (Schmidt et al. 1985). Similar tissue taken from normal mice has been used as a model in the present work to investigate how such a pattern of convergent migration might take place. Tissue treated cytochemically to reveal differences in surface alkaline phosphatase activity enabled a number of cell surface area measurements to be carried out by image analysis. The position where these measurements had been taken was then related to the geometrical centre of the tissue. Epithelial cell surface area halved during migration from edge to centre of these disc-like structures. An equation used to describe the positional dependence of this change had the form rho(r) = 6.86r(-0.24), where rho is cell density and r is cell distance from the centre measured in micrometers. Further calculation showed this decrease to be insufficient to explain all features associated with convergent migration. Differential changes occurring in the rate of cell migration and premature cell loss were also considered as possible factors affecting migration patterns. Mathematical modelling of these variables produced a migration pattern consistent with previous experimental findings. This model assumes that half the cell population is lost during migration and that cell migration rate near the centre is four times greater than at the edge. It should be possible to test this latter prediction in future experiments.

Alkaline Phosphatase↗
PubMed1993

Detection of two forms of GP330. Their role in Heymann nephritis.

Heymann nephritis is characterized by glomerular immune deposits that contain a glycoprotein called gp330. The deposits are believed to result from shedding of immune complexes formed on podocytes. Complexes are also shed from proximal tubule cells, when antibodies combine with gp330 on the cell surface. We performed the present study to investigate what portion of the gp330 molecule is shed, using a rabbit antiserum against a peptide deduced to be in the cytoplasmic domain of gp330, as well as a rabbit antiserum and two monoclonal antibodies that recognize extracellular epitopes of gp330. The anti-cytoplasmic peptide antiserum precipitated from Fx1A (a crude renal cortical membrane preparation), a protein with a mass of about 440 kd that was reactive with two monoclonal anti-gp330 antibodies. (In our experiments, the protein called gp330 generally has a mass estimated to be about 440 kd.) The anti-cytoplasmic peptide antiserum also reacted with a truncated gp330 protein produced in transfected COS cells. Immunohistochemical studies showed that all the antibodies recognized the same group of epithelial cells. However, as seen in immunoultrastructural studies of proximal tubules, the anti-cytoplasmic peptide antiserum reacted only with components at the base of microvilli, whereas the anti-gp330 ectodomain antibodies identified material not only at the base, but over the surface of microvilli as well. In rats with Heymann nephritis, glomerular deposits and material shed into tubule lumens reacted with antibodies against extracellular epitopes of gp330, but not with the anti-cytoplasmic peptide antiserum. We propose that there are two forms of gp330 on the cell surface of proximal renal tubules. One form is restricted to coated pit regions at the base of microvilli and has a cytoplasmic domain containing a sequence deduced from a partial complementary DNA encoding gp330. The other form is present over microvilli (and possibly at the base of microvilli as well) and lacks the cytoplasmic domain deduced from the complementary DNA. The complexes that are shed in Heymann nephritis contain either a portion of gp330 cleaved from the full-length molecule or a form of gp330 that lacks the cytoplasmic domain.

Amino Acid Sequence↗
PubMed1993

A re-evaluation of radical total glossectomy.

Total glossectomy, with or without accompanying total laryngectomy, in many respects represents the radical extreme of the treatment spectrum for oral cavity/oropharyngeal carcinoma. It necessarily impacts greatly upon the functions of speech and deglutition and thereby upon the patient's quality of life. The candidates for this surgical procedure are, however, already severely debilitated with respect to these functions due to the extent of their disease and tumor-induced pain. When considering the alternatives, radical surgery becomes more acceptable, for, at the very least, it offers substantial palliation. This article reviews the current literature regarding the procedure itself, potential outcomes and presents a small case series of reconstructive options.

Aged↗
PubMed1993

Recombinant alpha 2B interferon (IFN) in the treatment of chronic hepatitis C disease in thalassaemia major (TM).

Hepatitis C virus (HCV) is responsible for the majority of cases of post transfusion non-A non-B (NANB) hepatitis in thalassaemia major (TM). Fifteen multi-transfused TM patients with serological, biochemical, histological and molecular biological evidence of HCV infection have been treated for six months with recombinant alpha interferon (IFN). Eleven (73%) responded, 8 (53%) had complete response (CR), 3 (20%) partial response (PR) and 4 (27%) did not respond (NR) to IFN. Natural killer (NK) cell activity 24 hours after the first dose of IFN was significantly increased in responders as compared to non-responders. Liver histology showed an overall reduction of portal inflammation and periportal necrosis in the responding patients. HCV RNA disappeared from serum in 8 (15) responders and partial responders. Non responders remained positive. HCV RNA was tested and found to be positive in liver tissue material in 7 patients, five of those were re-tested after IFN treatment. Two became negative (both CR) 3 remained positive despite biochemical response to IFN. The degree of induction of peripheral blood mononuclear cell 2'5' oligoadenylate synthetase messenger RNA (2-5 OAS mRNA), an enzyme induced by IFN, after the first dose of IFN did not correlate with response neither was any significant interaction with cytokines observed; tumour necrosis factor (TNF), interleukin-1. (IL-1) and CD4:CD8 ratios did not change. We conclude that IFN should be given to all TM patients with chronic active hepatitis due to HCV.

Adolescent↗
PubMed1993

Structural analysis of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor.

The recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor ([3-153]M-CSF) consists of 302 amino acid residues and has a molecular mass of about 32 kDa, as estimated by SDS-PAGE. Two covalently linked subunits constitute a bioactive homodimer. The structure of the purified protein, expressed in Escherichia coli and refolded from inclusion bodies, was studied. The amino acid sequence was determined by automated Edman degradation of fragments obtained from degradation with CNBr and iodosobenzoic acid as well as by digestion with Glu-C endopeptidase of reduced and alkylated M-CSF. The absence of free thiol groups in the molecule was confirmed with Ellman reagent, which indicated the presence of seven disulfide linkages per homodimer. Sequence analysis of cystine-containing peptides, identified by comparing the peptide maps from unmodified and performic acid-oxidized pepsin digests, gave the following results. (1) Six out of seven disulfide linkages were formed between Cys 7 and Cys 90, Cys 48 and Cys 139, and Cys 102 and Cys 146 at each pair of positions as either intra- or inter-chain disulfides. (2) The remaining disulfide linkage linked Cys 31 of one subunit to Cys 31 of the second subunit of M-CSF. Based on our findings, a two-dimensional model is proposed in which the possible covalent linkage is suggested between the two subunits of the bioactive [3-153]M-CSF molecule.

Amino Acid Sequence↗