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Biomedical subjects

T Gilbert

Publications and source records attributed to T Gilbert.

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

Defect of nephrogenesis induced by gentamicin in rat metanephric organ culture.

BACKGROUND: In the rat, in utero exposure to gentamicin during early renal differentiation leads to a permanent nephron deficit. The aim of the present study was to analyze, in vitro, the potential direct effect of gentamicin on early nephrogenesis. EXPERIMENTAL DESIGN: We used paired rat metanephric organ cultures from 14 (F14) or 15-day-old (F15) fetuses. We measured gentamicin accumulation into explanted metanephroi and then assessed in vitro growth in the absence or presence of the drug. Glomerular labeling and counting were performed on the whole explant to analyze the effect of antibiotics on early nephrogenesis. RESULTS: Growth of F14 metanephric explants in the presence of 50 micrograms of gentamicin/ml was significantly reduced from 4 days onwards as compared to controls, whereas F15 explants grown with gentamicin displayed a normal in vitro development. After 6 days of culture, F14 and F15 explants had the same accumulation of gentamicin (1 microgram/mg protein) but the gentamicin content was 4 times larger in F15 explants. At both ages, gentamicin-exposed metanephric explants exhibited a significant reduction in their number of nephrons. However, the effects of 50 micrograms of gentamicin/ml on nephrogenesis were significantly more drastic on F14 than F15 explants (35% versus 18%). When grown with 0.5 microgram of gentamicin/ml, F14 explants still exhibited a 16% defect in nephrogenesis as compared with controls, and about the same reduction was observed for cultures in the presence of 100 micrograms/ml of streptomycin and 100 IU/ml of penicillin. Incubation of F14 explants with streptomycin alone for 6 days had no effect on nephrogenesis. CONCLUSIONS: These results indicate that gentamicin induces a significant reduction in the number of nephrons in metanephric explants and that this effect is more important on less differentiated metanephroi. Metanephric organ culture combined with glomerular labeling represents a useful model to test the effect of various growth factors and other drugs on early nephrogenesis.

Animals↗
PubMed1993

Learning disability nursing: from normalization to materialism--towards a new paradigm.

For almost 2 decades the 'normalization principle' has provided the dominant paradigm for the planning and evaluation of nursing services to people with a learning disability within the United Kingdom. This has reflected the international influence of this philosophy in services to a wide range of disadvantaged and marginalized groups. However, this paper will argue that nurses working with this client group are facing a range of social and political changes which expose the weaknesses in this paradigm. This calls for a new approach to the way the relationship between practitioners and their clients is conceptualized. Here it is argued that a materialist paradigm provides a more realistic framework upon which to base future nursing practice.

Culture↗
PubMed1993

Isolation of glucagon antagonists by random molecular mutagenesis and screening.

Glucagon has an important role in the regulation of glucose homeostasis, and glucagon antagonists may be effective therapeutic agents in the control of diabetes mellitus. We were able to identify a number of analogs with antagonist activity by creating libraries of mutant glucagon coding sequences, expressing them in a yeast (Saccharomyces cerevisiae) secretion system, and screening for clones that produce analogs that inhibit the glucagon stimulation of rat hepatocyte membrane adenylate cyclase. These libraries were constructed by allowing random misincorporation during the synthesis of oligonucleotides that contained the complete coding sequence for mammalian glucagon or for an analog (desHis1-glucagon) that had partial antagonist activity. We developed and used a simplified screening assay to test culture broths from > 3500 individual transformant yeast clones for their ability to inhibit glucagon-dependent adenylate cyclase activity. Ultimately, > 20 different analogs with antagonist activity were identified by recovering and sequencing plasmid DNA from yeast strains that were positive in the screening assay. Interestingly, several analogs were identified repeatedly in independent yeast clones and certain amino acid substitutions occurred in more than one analog. This clustering of randomly isolated mutations clearly delineates the regions of the glucagon molecule that are important for designing improved glucagon antagonists. A subset of the antagonists identified in yeast broth were produced by peptide synthesis to confirm their activities as pure compounds.

Adenylyl Cyclases↗
PubMed1992

Cloning and expression of a cellular high density lipoprotein-binding protein that is up-regulated by cholesterol loading of cells.

Plasma membranes of cultured cells contain high affinity receptors for high density lipoprotein (HDL) that appear to mediate removal of excess intracellular cholesterol. Recent studies using ligand blot analysis have identified a 110-kDa membrane protein which has features predicted for an HDL receptor, in that it preferentially binds HDL apolipoproteins and undergoes up-regulation in response to cholesterol loading of cells. In this study, we isolated a cDNA clone from an expression library using an antibody raised against partially purified 110-kDa HDL-binding protein. This clone encodes a novel cell protein, designated HBP, comprised mostly of 14 imperfect tandem repeats of approximately 70 amino acids in length. Each repeat appears to contain two amphipathic helices. Expression of HBP in cultured cells was increased severalfold when cells were loaded with cholesterol, as evident by increases in both HBP mRNA and membrane-associated protein. Overexpression of HBP in mammalian cell transfectants was associated with higher HDL binding to isolated cell protein and with modest increases in HDL binding to the cell surface. Proteins identified by ligand blot analysis had lower apparent M(r) than the primary HBP gene product and varied in M(r) and in HDL binding activity between cell types, suggesting that HBP undergoes cell-specific processing. These results provide preliminary evidence that HBP is a component of a cellular pathway that facilitates removal of excess cholesterol from cells, perhaps through its interaction with HDL. However, the predicted structure of HBP does not conform to that of any known receptor, suggesting that it does not function as a classic plasma membrane receptor.

Amino Acid Sequence↗
PubMed1991

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells.

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.

Alkaline Phosphatase↗
PubMed1991

Long-term effects of mild oligonephronia induced in utero by gentamicin in the rat.

Renal clearance studies and morphologic observations were performed in rats aged 14, 21, and 28 d and 3, 6, 9, and 12 mo born with a 20% reduction in nephron number after administration of 75 mg/kg/d of gentamicin to their mothers during the second half of gestation. Gentamicin was still present in urine 3 mo after birth. Morphologic damage characteristic of gentamicin accumulation was observed in the kidney on d 14 and 21. Adequate compensatory adaptation to oligonephronia occurred for glomerular function within 14 d of birth, but tubular phosphate reabsorption was significantly low on d 21. On d 28, no evidence of histologic or functional damage to the kidney was observed. At 3 mo, mesangial lesions were observed in rats of the gentamicin group, whereas they were rarely present in 6-mo-old control rats. Furthermore, glomerular sclerotic lesions were already evident in about 5% of the juxtamedullary nephrons. The same percentage of injured nephrons was not observed before 12 mo in controls. Complementary morphologic data obtained in 24-mo-old rats showed that glomerulosclerosis involved 40% of the juxtamedullary nephron population at this age in animals of the gentamicin group versus 21% in controls. It is concluded that in the young rats born with oligonephronia of gentamicin-treated mothers neither the gentamicin remaining in the kidney cells nor the injuries it caused them prevented compensatory adaptation of the kidney to a reduced number of nephrons. However, although this oligonephronia was mild, it might have been sufficient to cause early development of glomerular sclerosis in the adults.

Abnormalities, Drug-Induced↗
PubMed1991

Induction of vacuolar apical compartments in the Caco-2 intestinal epithelial cell line.

Complete disruption of the microtubular network by colchicine or nocodazole in Caco-2 intestinal epithelial cells results in the appearance of basolateral microvilli and brush border-containing intracellular vacuoles (vacuolar apical compartment: VAC). These vacuoles are surrounded by a terminal web, express apical markers and exclude basolateral markers. The vacuoles do not originate from internalized apical or basolateral plasma membrane and their development is blocked by protein synthesis inhibitors, suggesting that they are newly synthesized. After removal of the microtubule inhibitors, VACs are usually degraded and/or released into the lateral intercellular space. Rarely was fusion with the apical membrane observed. These experiments support a role for microtubules in the biogenesis of the apical surface and indicate that, under some conditions, apical plasma membrane assembly may occur in the cytoplasm, as observed in some human pathological states.

Cell Line↗
PubMed1990

A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting.

Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.

Amino Acid Sequence↗
PubMed1990

Expression, purification, and characterization of human factor XIII in Saccharomyces cerevisiae.

Factor XIII is the terminal enzyme of the clotting cascade. A cDNA sequence encoding human placental factor XIII was expressed in Saccharomyces cerevisiae with the yeast ADH2-4c promoter. Expression levels were a strong function of the noncoding flanking DNA content of the construction. When the terminal 3'-flanking noncoding DNA was removed, expression increased approximately 50-fold. The protein was produced in quantity by high-yield fermentation and purified to homogeneity. The recombinant protein was cleaved by thrombin at the same activation site as purified human placental FXIII and exhibited 100% enzymatic activity. At high thrombin concentrations rFXIIIa was cleaved into inactive 54- and 25-kDa polypeptides. The identity of these cleavage sites and the blocked N-terminus to that of the human protein was revealed by amino acid microsequencing. A time course of thrombin activation was performed and the relative distribution of the thrombin-cleaved subunits to the uncleaved zymogen subunits determined; the results were consistent with the half of the sites catalytic model for transglutaminase activity proposed by Chung et al. (Chung, S. I., Lewis, M. S., & Folk, J. E. (1974) J. Biol. Chem. 249, 940-950, 1974) and Hornyak et al. (Hornyak, T. J., Bishop, P. D., & Shafer, J. A. (1989) Biochemistry 28, 7326-7332). Equilibrium and velocity sedimentation analysis indicated that rFXIII exists as a 166-kDa nondissociating dimer that behaves as a compact particle of 8.02 S. Thus, all of the properties of rFXIII thus far examined are consistent with those reported for human platelet and placental FXIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1990

Immediate and long-term renal effects of fetal exposure to gentamicin.

Aminoglycoside antibiotics, like gentamicin, given to pregnant females cross the placenta and accumulate in the fetal kidney, which, like the adult kidney, was found to be the major site of deposition. In young guinea-pigs whose mothers were given gentamicin during the week following nephrogenesis in the fetus, nephron growth was found to be retarded temporarily. In rats whose mothers were given gentamicin during the period of fetal nephrogenesis, the final number of nephrons was reduced by about 20%. In both cases, renal development was impaired, although the concentration of gentamicin in the fetal kidney was lower than that measured in the kidney of human fetuses whose mothers had received a single injection of aminoglycoside. In rats exposed to gentamicin in utero, cellular damage of the undifferentiated and differentiating renal tissues was observed. It is, there are likely that the oligonephronia observed in animals born of gentamicin-treated mothers resulted from a direct effect of the drug at early stages of nephrogenesis. When gentamicin administration to the mother was prolonged, part of the oligonephronia observed at birth might have also resulted from fetal growth retardation, secondary to adverse effects of the drug on the mother. Providing it was not associated with fetal growth retardation, the presence of high gentamicin concentrations in the fetal kidney at late stages of nephrogenesis did not affect nephron differentiation. Long-term studies of rats born with gentamicin-induced oligonephronia showed that neither the antibiotic still present in kidney several weeks after birth, nor the injuries it caused, prevented renal growth and morphological adaptation of the nephrons to their reduced number.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1990

Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells.

To characterize the mechanisms that determine the apical polarity of proteins anchored by glycosylphosphatidylinositol (GPI), we studied the targeting of a GPI-anchored form of a herpes simplex glycoprotein, gD-1, in transfected MDCK cells. Using a biotin-based targeting assay, we found that GPI-anchored gD-1 was sorted intracellularly and delivered directly to the apical surface. Endocytosis of GPI-anchored gD-1 occurred slowly and preferentially from the apical domain, while transcytosis of the basolateral fraction did not occur at a significant rate (incompatible with being a precursor to the apical pool). Prevention of tight junction formation by incubation in medium with micromolar Ca2+ resulted in expression of GPI-anchored gD-1 on the free surface, but not on the attached surface of the cell. Our results indicate that the apical polarity of a GPI-anchored protein is generated by vectorial delivery to the apical membrane, where its distribution is maintained by slow endocytosis and by a retention system not necessarily involving the tight junction.

Animals↗
PubMed1990

Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences.

Rat 6 fibroblast cell lines expressing wild-type chicken liver glycoprotein receptor (CHL) or chimeric receptors with alternate cytoplasmic tails were produced to study the role of the cytoplasmic tail in mediating receptor localization in coated pits and endocytosis of ligand. Cells expressing CHL or cells expressing a hybrid receptor that contains the cytoplasmic tail of the asialoglycoprotein receptor display high-efficiency endocytosis of N-acetylglucosamine-conjugated bovine serum albumin in experiments designed to measure an initial internalization step, as well as in studies of continuous uptake and degradation. Substitution of the cytoplasmic tail by the equivalent domain of rat Na,K-ATPase beta subunit or by a stretch of Xenopus laevis globin beta chain does not abolish endocytosis but decreases the endocytosis rate constant from 15%-16%/min to 2.4% and 6.5%/min, respectively. Electron microscopy was used to visualize the glycoprotein binding sites at the surface of Rat 6 cells transfected with the various receptors. The percentage of receptors found in coated areas ranged from 32% for CHL to 9% for the Na,K-ATPase hybrid, indicating that clustering in coated pits correlates with efficiency of endocytosis. We concluded that replacement of the CHL cytoplasmic tail with unrelated sequences does not prevent, but decreases to varying extents, coated-pit localization and endocytosis efficiency. The construct with NH2-terminal globin tail lacks a signal for high-efficiency localization in coated pits but nevertheless is directed to the pits by an alternative mechanism.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes.

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.

Amino Acid Sequence↗
PubMed1989

Development of a DNA probe from the deoxyribonucleotide sequence of a 3-N-aminoglycoside acetyltransferase [AAC(3)-I] resistance gene.

The aacC1 gene encoding the 3-N-aminoglycoside acetyltransferase [AAC(3)-I] was cloned from enteric plasmid pJR88, and its deoxyribonucleotide sequence was determined. Significant nucleotide homology was noted in the region extending from the proposed -35 sequences through the first 59 base pairs of the aacC1 gene open reading frame (ORF) and the upstream flanking regions and ORFs of several other antibiotic resistance genes. Sequences were noted to be homologous with the 6'-N-aminoglycoside acetyltransferase [AAC(6')-I], 2''-O-aminoglycoside adenylyltransferase [AAD(2'')], and 3''-O-aminoglycoside adenylyltransferase [AAD(3'')] resistance genes; the OXA-1, OXA-2, and PSE-2 beta-lactamase genes; and several dihydrofolate reductase genes. Small regions of homology were noted in the 3'-flanking regions of these resistance genes as well. A DNA probe for the aacC1 gene was selected from the nucleotide sequence information and was tested against a series of genetically and enzymatically defined strains. The probe, which proved specific for the aacC1 gene, was then tested against a series of 58 gentamicin-susceptible and 219 gentamicin-resistant gram-negative bacilli isolated from patients at the Seattle Veterans Administration Medical Center. Only six clinical isolates were noted to carry the aacC1 gene. Each was resistant to gentamicin but susceptible to kanamycin, tobramycin, and amikacin. The presence of homologous regions of DNA at both the 3' and 5' ends of the aacC1 gene reinforces the importance of choosing probes from within the ORFs of genes and of avoiding flanking sequences. When the homology with other sequences extends into the ORF, as it does with the aacC1 gene, development of a specific probe may require determination of the nucleotide sequence.

Acetyltransferases↗
PubMed1989

Characterization of a human-mouse chimeric antibody reactive with a human melanoma associated antigen.

The data presented demonstrate that a human-murine chimeric antibody has been generated that retains the immunoreactivity and has similar pharmacokinetic properties of its parent murine monoclonal antibody NRML-05. A competitive ELISA assay demonstrated that antigen reactivity of both parent and chimera were nearly identical. In a direct cell binding assay, NRML-05 and chimeric antibodies were immunoreactive, 81% and 83%, respectively. Scatchard Analysis of the antibodies indicate very similar affinities for NRML-05 (4.4 x 10(9) M-1) and chimera (2.7 x 10(9) M-1). Localization of the two antibodies to tumor xenografts in nude mice were very similar in biodistribution studies. The chimeric antibody is not as well recognized by antiglobulin from patients who have responded to non-idiotypic murine antibody determinants. Although this data does not predict how immunogenic a chimera would be in the clinical setting, it does suggest that patients without significant anti-idiotypic antiglobulin response could benefit from second and subsequent administrations with chimeric antibody. Even though anti-idiotypic responses may occur with the chimera, these may be reduced as a result of the presentation of these epitopes on the less immunogenic human constant domains.

Animals↗