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J H Todd

Publications and source records attributed to J H Todd.

At least 19 recordsLinked to original sources
PubMed1998

Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA.

The organization of the human metallothionein (MT) gene family is more complex than the commonly used mouse and rat models. The human MTs are encoded by a family of genes consisting of 10 functional and 7 nonfunctional MT isoforms. One objective of this study was to determine if the accumulation of MT protein in cultures of human proximal tubule (HPT) cells exposed to metals is similar to that expected from the knowledge base obtained from rodent models. To accomplish this objective, HPT cells were exposed to both lethal and sublethal concentrations of Cd2+, Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ and MT protein levels were determined. The results were in general agreement with animal model studies, although there were some exceptions, mainly in areas where the animal model database was limited. In clear agreement with animal models, Cd2+, Zn2+, and Cu2+ were demonstrated to be potent inducers of MT protein accumulation. In contrast to the similarity in MT protein expression, we obtained evidence that the human renal MT-2 gene has a unique pattern of regulation compared to both animal models and human-derived cell cultures. In the present study, we determined that MT-2A mRNA was not induced by exposure of HPT cells to Cd2+ or the other metals, a finding in contrast to studies in both animal models and other human cell culture systems in which a high level of MT-2 mRNA induction occurs upon exposure to Cd2+ or Zn2+. While MT protein expression may be similar between humans and animal models, this finding provides initial evidence that regulation of the genes underlying MT protein expression may be divergent between species.

Aged↗
PubMed1998

Differential expression of human metallothionein isoform I mRNA in human proximal tubule cells exposed to metals.

In contrast to the single metallothionein (MT)-1 gene of the mouse, the human MT-1 gene family is composed of seven active genes and six pseudogenes. In this study, the expression of mRNA representing the seven active human MT-1 genes was determined in cultured human proximal tubule (HPT) cells under basal conditions and after exposure to the metals Cd2+, Zn2+, Cu2+, Hg2+, Ag2+, and Pb2+. Basal expression of MT-1X and MT-1E mRNA in HPT cells was similar to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. In contrast, mRNAs representing the basal expression of MT-1A and MT-1F were a minor transcript in HPT cells. Treatment of HPT cells with Cd2+, Zn2+, or Cu2+ increased the levels of MT-1E and MT-1A mRNA, but not the levels of MT-1X or MT-1F mRNA. The increase in MT-1E mRNA appeared to be influenced mainly by exposure to the various metals, whereas the increase in MT-1A mRNA was influenced more by exposure to a metal concentration eliciting a loss of cell viability. Treatment of HPT cells with the metals Hg2+, Ag2+, and Pb2+ was found to have no effect on the level of MT-1 mRNA at either sublethal or lethal concentrations. Using HPT cells as a model, these results suggest that new features of MT gene expression have been acquired in the human due to the duplication of the MT-1 gene.

Cells, Cultured↗
PubMed1998

Differential expression of the MT-1E gene in estrogen-receptor-positive and -negative human breast cancer cell lines.

The goal of this study was to determine which of the 10 functional metallothionein (MT) genes are expressed in four human breast cancer cell lines and whether expression varies among the cell lines. Using reverse transcription polymerase chain reaction (RT-PCR) technology, it was shown that there was no expression of mRNA for the MT-1A, MT-1B, MT-1F, MT-1G, MT-1H, MT-3, and MT-4 genes in any of the four cell lines. All four cell lines were shown to express mRNA for the MT-2A and MT-1X genes. The expression level of mRNA for the MT-2A gene demonstrated modest differences among the cell lines, whereas expression of the MT-1X gene was consistent. In contrast, mRNA for the MT-1E gene was expressed in only two of the four cell lines and expression correlated to the estrogen receptor status of the cell lines. The two estrogen-receptor-positive cell lines showed no mRNA expression for the MT-1E gene. In the two estrogen-receptor-negative cell lines, mRNA expression for the MT-1E gene was elevated with expression levels similar to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The cellular content of MT protein was also shown to be elevated in the estrogen-receptor-negative cell lines that express MT-1E mRNA. These results suggest a possible relationship between estrogen receptor status and MT-1E gene expression in human breast cancer.

Breast Neoplasms↗
PubMed1997

Expression of MT-3 mRNA in human kidney, proximal tubule cell cultures, and renal cell carcinoma.

The human metallothionein 3 (MT-3) gene has recently been identified and characterized as a brain-specific MT having growth inhibitory activity for neuronal cells. One objective of the present study was to determine if MT-3 is brain-specific or also present in the renal system, a site for chronic toxicity due to heavy metal exposure. Using RT-PCR methodology, MT-3 mRNA was shown to be expressed in the human renal system at levels below mRNA for the beta-actin gene. MT-3 mRNA was shown to be expressed in all samples obtained from both the developing and adult renal systems, from 20 weeks of fetal age to 72 years. Cultures of human proximal tubule (HPT) cells were used to determine if MT-3 mRNA expression is influenced by metal exposure. Exposure of HPT cells to either Zn2+ or Cd2+ resulted in an early (within 24 h), but unsustained increase in MT-3 mRNA. The demonstration of MT-3 mRNA expression in the kidney indicates that MT-3 may play an important early role in the response of the cell to metal exposure. MT-3 mRNA expression was also examined in tissues and cells from three cases of renal cell carcinoma. MT-3 was found to be expressed in all three cases at levels similar to those found for normal kidney, providing evidence that MT-3 mRNA expression is not altered in this cancer.

Adolescent↗
PubMed1997

Poly-l-aspartic acid protects cultured human proximal tubule cells against aminoglycoside-induced electrophysiological alterations.

Cultured human proximal tubule cell monolayers maintained on permeable supports were treated simultaneously with the aminoglycoside antibiotic, gentamicin, and poly-L-aspartic acid (PAA), an inhibitor of aminoglycoside nephrotoxicity. Following 4 days of exposure, cell monolayers were placed into Ussing chambers to allow monitoring of transepithelial electrical properties. For each of the three cell isolates examined, aminoglycoside-induced alterations in electrogenic transport, reflected by changes in short-circuit current (Isc), as well as alterations in paracellular properties, indicated by changes in transepithelial electrical resistance (RT), were diminished in the presence of PAA. Alterations resulting from selective basolateral exposure to gentamicin were unchanged in the case of apically applied PAA and attenuated only when PAA acid was added basolaterally. This is the first demonstration of PAA inhibition of aminoglycoside-induced cellular alterations involving human cells.

Anti-Bacterial Agents↗
PubMed1997

Expression of hsc 70, but not hsp 70, in human third molar dental pulp.

The constitutive (hsc 70) and inducible (hsp 70) isoforms of heat shock protein 70 are important members of the superfamily of stress related proteins that protect and promote the recovery of cells from physiological and pathologic stress. The goal of this study was to define the baseline expression of hsc 70 and hsp 70 in disease-free, minimally stressed human dental pulp of the adult 3rd molar. Immunolocalization demonstrated moderate to heavy staining intensity for hsc 70 in both the cytoplasm and nucleus of odontoblasts and fibroblasts comprising the human pulp. Endothelial and smooth muscle cells displayed weak to moderate immunoreactivity for hsc 70 in both the cytoplasm and nucleus. Schwann cells demonstrated only weak nuclear staining for hsc 70. No immunoreactivity for hsp 70 was observed in any cell type in human pulp. Western, northern, and RT-PCR analysis of pulp preparations confirmed the expression of hsc 70 mRNA and protein within components of the pulp. These results demonstrate that cells of the human pulp express, under conditions of minimal stimulation, a key component of the stress response protein superfamily. The expression of hsc 70 under conditions of minimal stress may provide pulp components an advantage in resisting cell injury when stress occurs.

Adult↗
PubMed1996

Expression of heat shock protein 27 in developing and adult human kidney.

The expression of heat shock protein (hsp) 27 was examined in the developing and adult human kidney. Immunolocalization using a monoclonal antibody against human hsp 27 demonstrated immunoreactivity in both the developing and adult kidney. Low to moderate immunoreactivity for hsp 27 was observed in the fetal and adult proximal tubule, distal tubule, and mesangial cells of the glomeruli. Intense immunoreactivity for hsp 27 was localized to the cortical and medullary collecting ducts in both the adult and fetal kidney, with the most intense staining in the medullary regions. The loop of Henle demonstrated no immunoreactivity for hsp 27. The blastemal element of the developing kidney showed no hsp immunostaining and the ureteric bud demonstrated moderate staining. Western, northern, and reverse transcription-polymerase chain reaction (RT-PCR) analyses disclosed no significant differences in hsp 27 mRNA or protein level as a function of gestational age. An analysis of the phosphorylation state of hsp 27 showed the majority of hsp 27 to be present in the unphosphorylated isoform for both adult and fetal samples. These studies are the first to demonstrate the presence of hsp 27 in the human kidney. It is suggested that this pool of hsp 27 is constitutive as it appears in an inactivated state; localized to the cytoplasm and in an unphosphorylated state.

Adolescent↗
PubMed1996

Isoform-specific expression of metallothionein mRNA in the developing and adult human kidney.

The organization of the metallothionein (MT) gene family has been demonstrated to be much more complex in humans than in the mouse, and possibly rodents in general. For humans, the MTs are encoded by a family of genes located at 16q13 representing 10 functional and 7 non-functional MT isoforms. In the present study, the 5' and 3' untranslated region sequences of the highly conserved, functional MT genes were utilized to generate primer pairs for the analysis of isoform-specific MT mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR). Human kidneys from 13 weeks gestation through adulthood were examined for the expression of MT protein and mRNA. Immunohistochemical analysis demonstrated MT immunoreactivity to be confined exclusively to the proximal tubules of the adult and developing kidney. For all MT-positive cells, MT was localized in the cytoplasm and nuclear localization was variable. There was no correlation between nuclear staining and stage of development. Of the 10 MT genes examined (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, MT-3, and MT-4), mRNAs representing the MT-1E, MT-1F, MT-1X, and MT-2A genes were consistently expressed in all samples regardless of gestational age. There was no indication of a 'fetal form' of MT analogous to that noted to occur in human liver. Messenger RNA for the MT-1A gene was detected in 2 of 6 renal samples without correlation to gestational age. In no instance was mRNA for the MT-1B, MT-1G, MT-1H, MT-3 or MT-4 genes detected. These studies detail the initial determination of MT gene expression in the human renal system and provide the PCR primers for testing and determination of MT gene expression in other organ systems.

Adolescent↗
PubMed1996

Expression of heat shock protein 27 in adult human third molar dental pulp.

This study is the first to define the expression of hsp 27 in the pulp of the adult human third molar. Using a monoclonal antibody against human hsp 27, immunoreactivity was demonstrated in the odontoblasts, odontoblast processes, pulp fibroblasts, and smooth muscle and endothelial cells of vessel walls. Nerves were negative. Pulp fibroblasts were characterized by cytoplasmic staining and variable nuclear staining. Odontoblasts also displayed consistent cytoplasmic staining and variable nuclear staining. Western, Northern, and RT-PCR analysis confirmed the expression of hsp 27 mRNA and protein. Hsp 27 was also shown to be present in both the unphosphorylated and phosphorylated isoforms. In general, nuclear localization and phosphorylation of hsp 27 has been correlated with cells responding to stress or other stimuli. This study demonstrates that pulp from a single human third molar provides sufficient material to support a detailed molecular analysis of gene expression.

Adult↗
PubMed1995

Renal brush border membrane vesicle aminoglycoside binding and nephrotoxicity.

The in vitro binding affinity (KD) of aminoglycoside (AG) antibiotics to renal brush border membranes (BBM) has previously been correlated with several variables (sex, age and toxic potential of specific antibiotics) influencing in vivo AG nephrotoxicity in rats. The initial intent of our study was to extend this correlation to the in vivo difference in sensitivity to tobramycin recently observed between two strains of rats. However, the tobramycin binding affinity (KD) failed to correlate with the in vivo strain difference in AG nephrotoxicity. In addition, a comparison of BBM vesicle tobramycin binding affinity between sexes failed to correlate with an earlier report of in vivo gender differences in tobramycin nephrotoxicity. Comparison of binding capacities for male Sprague-Dawley rats and male Fischer rats yielded the only instance where binding parameters correlated with the reports of in vivo nephrotoxicity comparisons. Preliminary examinations of tobramycin binding using renal BBM vesicles derived from a single human kidney revealed binding kinetics and characteristics similar to rat BBM binding. Tobramycin binding was rapid and saturable for each type of BBM vesicle preparation. Scatchard analyses indicated low affinity, high capacity binding characteristics, as well as a single binding site in each case. Polyaspartic acid, which blocks in vitro BBM tobramycin binding as well as in vivo nephrotoxicity in rats, also blocked binding of tobramycin to human BBM vesicles. These data indicate that AG binding is qualitatively similar for rat and human BBM vesicle preparations. However, examination of the quantitative kinetic aspects of AG binding to BBM vesicles (KD and binding capacities) suggests that these parameters may not be critical determinants in the pathogenesis of Ag nephrotoxicity.

Aminoglycosides↗
PubMed1994

Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age.

Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn. The growth medium utilized was a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 supplemented with selenium (5 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained. Confluency was reached within 3 to 6 days. A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates. That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained. By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity. However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or "micro-domes" that would not be visible by light microscopy. Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy. However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions. It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.

Cells, Cultured↗
PubMed1994

Selective exposure of human proximal tubule cells to gentamicin provides evidence for a basolateral component of toxicity.

The goal of the present study was to determine if cultured human proximal tubule (HPT) cells could provide evidence for a basolateral component of gentamicin toxicity. Six isolates of HPT cells were grown on Millicell filters and exposed to gentamicin either apically, basolaterally, or both apically and basolaterally. Toxicity was determined by the release of lactate dehydrogenase into the growth media. The results clearly demonstrated that basolateral exposure and combined apical and basolateral exposure to gentamicin resulted in significant levels of cell toxicity. In contrast, apical exposure to gentamicin elicited only marginal toxicity. The transepithelial flux of gentamicin was shown to be the same in either the apical to basolateral or the basolateral to apical direction. A two-step mechanism of gentamicin toxicity is proposed in order to integrate basolateral toxicity with known features of aminoglycoside nephrotoxicity.

Adult↗
PubMed1993

Variation in the electrical properties of cultured human proximal tubule cells.

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD), short-circuit current (Isc), and transepithelial specific resistance (RT). These electrical parameters were used to determine the degree of heterogeneity among independent isolates of human proximal tubule cell cultures. Seventeen independent isolates of cells were assessed, totaling 260 individual determinations of spontaneous electrical properties. On average, these cell monolayers displayed an apical-negative PD of 1.5 +/- 0.1 mV, an Isc of 2.7 +/- 0.2 microA/cm2, and an RT of 480 +/- 19 ohms x cm2. Each independent cell isolate, however, displayed electrical values within a narrow range, in some cases allowing isolates to be distinguished from one another. The individual isolates were also assessed for Na-coupled glucose transport, Na+,K(+)-ATPase activity, cAMP stimulation by parathyroid hormone (PTH), forskolin stimulation of Isc, and ouabain inhibition. With the exception of a strong correlation between Na+,K(+)-ATPase activity and Isc, these parameters, in contrast to electrical properties, were found to be consistent and did not reveal distinctions among the isolates. HPT cell cultures seem to consistently retain important features of proximal tubule differentiation while maintaining the variability, as demonstrated by electrical properties, that might be expected of cells isolated from a random population.

Cells, Cultured↗
PubMed1993

Electrical and freeze-fracture analysis of the effects of ionic cadmium on cell membranes of human proximal tubule cells.

We previously reported that cell cultures of human proximal tubule (HPT) cells respond to ionic cadmium in a manner consistent with well-defined Cd(2+)-elicited responses reported for in vivo systems. However, one unique finding was that the transepithelial electrical resistance and tight junction sealing strands were altered as a result of Cd2+ exposure at micromolar concentrations. These alterations are reexamined in detail in the present report to determine whether the Cd(2+)-induced alterations are specific alterations in the tight junction structure or reflect a general alteration in the cell membrane. Exhaustive analysis of tight junction sealing strands demonstrated no significant alterations due to Cd2+ exposure, even at the concentration that elicited a significant reduction in transepithelial resistance. Further analysis of intramembrane particle distribution demonstrated a significant increase in apical intramembrane particles, indicating that Cd2+ exposure altered the characteristics of the apical cell membrane. Overall, the results were consistent with evidence of Cd(2+)-induced alteration in the apical cell membrane of the HPT cell.

Cadmium↗
PubMed1992

In situ freeze-fracture of monolayer cell cultures grown on a permeable support.

The growth of cultured epithelial cells on permeable supports allows increased cell differentiation and the assessment of a variety of transcellular and paracellular transport processes. The need to assess the corresponding ultrastructural characteristics of these cells under identical conditions prompted this laboratory to develop a reliable method for producing freeze-fracture replicas of these cultures. Sections of filter inserts with the cell-side facing up are placed between layers of polyvinyl alcohol with a strip of mylar positioned on the layer of polyvinyl alcohol. Following freezing, the monolayer is fractured by lifting the mylar strip from the assembly. The result is a consistent fracture of the apical membrane sufficient for analysis of tight junction sealing strands, microvilli distribution, and intramembranous particle (IMP) distribution between apical and lateral membrane domains. This method utilizes standard equipment and readily available materials and, most importantly, allows the freeze-fracture and replication of an undisturbed cell monolayer.

Cells, Cultured↗
PubMed1992

Aminoglycoside antibiotics alter the electrogenic transport properties of cultured human proximal tubule cells.

Monolayers of human proximal tubule (HPT) cells, when grown on permeable supports and mounted in Ussing chambers, spontaneously display a transepithelial potential difference (PD) and short-circuit current (Isc). These electrical parameters were used in the present study to determine if aminoglycoside exposure altered electrogenic sodium transport by HPT cells. The results of this determination demonstrated that exposure to gentamicin, at levels below that producing cell necrosis, caused a marked reduction in Isc and that this reduction followed the known in vivo nephrotoxicities of the aminoglycosides streptomycin, gentamicin, and neomycin. It was concluded through a similar analysis on a total of 14 isolates of HPT cells that the aminoglycosides repeatably reduced the electrogenic sodium transport of HPT cells. It was further determined that this alteration in electrogenic transport by gentamicin was mediated through exposure of the drug to the basolateral cell surface and that apical exposure had little effect. Evidence was obtained against the involvement of Na+, K(+)-ATPase, adenosine 3',5'-cyclic monophosphate, and sodium-coupled substrate transport in this alteration in electrogenic transport by the aminoglycosides. The basolaterally located Na+: CO3(-2):HCO3(-1) symporter is a possible site for aminoglycoside-induced nephrotoxicity.

Adult↗
PubMed1990

Expression of epidermal growth factor receptor in breast carcinoma.

A series of 90 patients with primary operable breast cancer and with up to 36 months of follow up were investigated for expression of epidermal growth factor receptor (EGFR) in their tumours by immunocytochemical staining with the monoclonal antibody EGFR 1. Tumour samples were snap frozen in liquid nitrogen immediately after resection and subsequently stained using a standard indirect immunocytochemical method. Tumour staining was assessed by two observers and scored on a four point scale (0-3). Thirteen (14%) tumours showed positive immunoreactivity. A strong correlation between distinct EGFR expression and short disease free interval was observed. Significant correlations were also shown with oestrogen and progesterone receptor expression and tumour nuclear size. No significant association was found with tumour size, lymph node stage, and histological grade. The association with disease free interval remained significant in multivariate analysis.

Antibodies, Monoclonal↗