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Biomedical subjects

G A Preston

Publications and source records attributed to G A Preston.

At least 19 recordsLinked to original sources
PubMed2001

Internalization of proteinase 3 is concomitant with endothelial cell apoptosis and internalization of myeloperoxidase with generation of intracellular oxidants.

The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory vascular diseases. We have determined whether granule proteins proteinase 3(PR3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, as examined by UV light, confocal, and electron microscopy. Coincident induction of apoptosis and/or the generation of intracellular oxidants were monitored. The results indicate that human endothelial cells (human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human lung microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epithelial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specific and different from that of MPO. Internalization of PR3, but not MPO, correlated with activation of apoptosis. Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the proteolytic activity of PR3 for the induction of apoptosis was examined by generating PR3-truncated fragments that did not contain the components of the catalytic triad. An apoptotic function was localized to the C-terminal portion of PR3. These studies reveal novel mechanisms by which the neutrophil granule proteins PR3 and MPO contribute to tissue injury at sites of inflammation.

Apoptosis↗
PubMed2000

Apoptotic response to growth factor deprivation involves cooperative interactions between c-Fos and p300.

Two preneoplastic cell lines have been utilized to study changes in the regulation of apoptosis during neoplastic progression [sup+I (stage I) and sup-II (stage II)]. Sup+I cells are prone to undergo apoptosis, while sup-II cells are relatively resistant. We report that induction of apoptosis in sup+I cells is tightly correlated with the formation of c-Fos/p300 complexes, which were not present in the non-apoptotic sup-II cells under the same conditions. When apoptosis was induced in the sup-II cells by over-expression of c-Fos, concomitant c-Fos:p300 complexes were detected. Over-expression of p300 resulted in apoptosis in sup-II cells and also in p53wt human tumor cells, but not in p53mutant human tumor cells. Over-expression of the C-terminal fragment of p300, which contains the c-Fos binding site, enhanced apoptosis, suggesting that the c-Fos:p300 complex is actively involved in apoptosis. We propose that p300 could function as a general mediator of transcription factor-induced apoptosis.

Animals↗
PubMed2000

ANCA antigens, proteinase 3 and myeloperoxidase, are not expressed in endothelial cells.

BACKGROUND: One hypothesis for the pathogenesis of vasculitis associated with antineutrophil cytoplasmic autoantibodies (ANCAs) proposes that ANCAs bind to ANCA antigens, such as proteinase 3 (PR3) or myeloperoxidase (MPO), which are produced by endothelial cells and expressed on their surfaces. There are conflicting reports, however, on whether endothelial cells express the ANCA antigen PR3, and there are no reports on endothelial expression of MPO. The aim of this study was to determine the presence or absence of PR3 and MPO mRNA in both venous and arterial endothelial cells, employing standard reverse transcription-polymerase chain reaction (RT-PCR) techniques and also the quantitative and highly specific method, TaqMan PCR. METHODS: RT-PCR (with 3 primer sets) and TaqMan PCR, a method for detecting low copy transcripts, were used to probe for PR3 and MPO transcripts in human endothelial cells from umbilical vein (HUVEC) and artery (HUAEC) and from lung microvascular (HLMVEC). Cells were treated with interferon-gamma (200 units/mL) or tumor necrosis factor-alpha (3 or 10 ng/mL) or both. RESULTS: Transcripts for PR3 and/or MPO were not detected in HUVEC, HUAEC, and HLMVEC by standard RT-PCR. Analyses for PR3 protein confirmed that PR3 is not expressed in HUVEC. HUVEC and HUAEC were negative for PR3 and MPO by TaqMan PCR. CONCLUSIONS: PR3 and MPO are not expressed in HUVEC, HUAEC, or HLMVEC. Endothelial cell presentation of endogenous PR3 and MPO antigens is not involved in the pathogenesis of ANCA-associated vasculitis. Alternative explanations need to be explored to determine the pathogenic effect of ANCAs.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1997

Effects of alterations in calcium homeostasis on apoptosis during neoplastic progression.

Our previous studies showed that early, stage I preneoplastic cells (sup+ I) are highly susceptible to apoptosis, whereas the later, stage II preneoplastic cells (sup- II) are relatively resistant. To examine possible mechanisms that might explain these differences in the regulation of apoptosis, Ca2+ homeostasis was analyzed and comparisons were made between these two Syrian hamster embryo cell lines. The Ca2+ indicator, fura-2, and fluorescent microscopy were used to measure intracellular free calcium concentrations, [Ca2+]i. The results indicated that the [Ca2+]i level in logarithmically growing sup+ I cells (approximately 100 nM) was considerably lower than that observed in sup- II cells (approximately 260 nM). Serum removal resulted in a reduction of [Ca2+]i in the sup+ I cells (approximately 82 nM), whereas the [Ca2+]i level in sup- II cells did not change. Endoplasmic reticulum (ER) calcium levels were determined by measuring thapsigargin-releasable Ca2+. Reduced ER calcium was consistently observed in cells induced to undergo apoptosis. Specifically, thapsigargin-releasable Ca2+ was greatly reduced in sup+ I cells (45 nM) as compared to sup- II cells (190 nNM) after 4 h in low serum. When sup- II cells were placed under conditions that resulted in apoptosis (thapsigargin or okadaic acid), decreased ER calcium was observed. To determine whether reduced ER calcium had a causative effect in apoptosis, ER calcium levels were exogenously increased in sup+ I cells by raising extracellular Ca2+ to 3 mM; ER calcium levels were maintained, and apoptosis was blocked. Studies were performed to determined whether the decrease in ER calcium could be attributed to reduced Ca2+ influx at the plasma membrane. To measure directly whether Ca2+ entry was decreased in sup+ I cells in 0.2% serum, Mn2+ uptake was used to monitor Ca2+ influx. The data show that in low serum, the rate of thapsigargin-induced Mn2+ entry in sup+ I cells was approximately 50% lower than that of sup- II cells, demonstrating that capacitative entry is reduced in sup+ I cells. In further support of this hypothesis, thapsigargin-treated sup+ I cells (0.2% serum) showed decreased Ca2+ entry upon raising extracellular Ca2+ from 0 to 2 mM. We report the novel finding that early preneoplastic cells, which exhibit a high propensity to undergo apoptosis, have decreased calcium entry at the plasma membrane, resulting in decreased ER calcium pools. This study provides new insight into mechanisms that can be involved in the regulation/dysregulation of apoptosis during neoplastic progression. Furthermore, the data imply that preneoplastic cells, which have developed a mechanism to maintain ER calcium, would be less susceptible to apoptosis and would thus have an increased potential for becoming transformed.

Animals↗
PubMed1996

Induction of apoptosis by c-Fos protein.

The role of c-Fos in apoptosis was examined in two Syrian hamster embryo cell lines (sup+I and sup-II) and a human colorectal carcinoma cell line (RKO), using the chimeric Fos-estrogen receptor fusion protein c-FosER. As previously reported, contrasting responses were observed when these two cell lines were placed under growth factor deprivation conditions; sup+I cells were highly susceptible to apoptosis, whereas sup-II cells were resistant. In this report, we show that the activated c-FosER protein induces apoptosis in sup-II preneoplastic cells in serum-free medium, indicating that c-Fos protein can induce apoptotic cell death in these cells. c-Fos-induced apoptosis was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that the c-Fos transcriptional activation activity is not involved. This conclusion was further supported by the observation that overexpression of v-Fos, which is highly proficient in transcriptional activation but deficient in the transcriptional repression activity associated with c-Fos, did not induce apoptosis. Constitutively expressed Bcl-2 delayed the onset of low-serum-induced apoptosis in sup+I cells and enhanced survival in sup-II cells. Further, coexpression of Bcl-2 and c-FosER in sup+I or sup-II cells protected the cells from c-FosER-induced apoptosis. The possibility that c-FosER-induced apoptosis requires a p53 function was examined. Colorectal carcinoma RKOp53+/+ cells, which do not normally undergo apoptosis in serum-free medium, showed apoptotic DNA fragmentation upon expression and activation of c-FosER. Further, when the wild-type p53 protein was diminished in the RKO cells by infection with the papillomavirus E6 gene, subsequent c-FosER-induced apoptosis was blocked. The data suggest that c-Fos protein plays a causal role in the activation of apoptosis in a p53-dependent manner. This activity does not require new protein synthesis and is blocked by overexpression of Bcl-2 protein.

Animals↗
PubMed1995

Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells.

Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of bee hives, was shown previously to block tumor promoter- and carcinogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechanisms of CAPE-induced differential cytotoxicity, nontumorigenic rat embryo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells (Wt3A) were used. As shown by nucleosomal-length DNA degradation, morphological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivariant flow cytometry, cell death induced by CAPE in the transformed Wt3A cells was apoptosis. Under the same CAPE treatment conditions, CREF cells transiently growth arrested. Both CREF and Wt3A cells were radioresistant, suggesting deficiencies in the proteins controlling the G1 checkpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the redox state of the cells. Depletion of cellular glutathione (GSH) with buthionine sulfoximine before CAPE treatment caused CREF sensitive to CAPE-induced cell death. GSH levels were also determined in CAPE-treated CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When the GSH levels were increased in Wt3A cells by treatment with the reducing agent, N-acetyl-cysteine before CAPE treatment, the Wt3A cells were partially rescued. Furthermore, Bcl2, which protects cells from oxidative stress, had a protective effect against CAPE-induced apoptosis in Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidant, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further proving that Wt3A cells were more sensitive than CREF cells to oxidative stress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determined by the loss of normal redox state regulation in transformed cells.

Acetylcysteine↗
PubMed1994

Regulation of apoptosis by low serum in cells of different stages of neoplastic progression: enhanced susceptibility after loss of a senescence gene and decreased susceptibility after loss of a tumor suppressor gene.

A cell culture model system has been used to study the susceptibility of cells to apoptotic cell death during different stages of neoplastic progression. This system consists of normal diploid Syrian hamster embryo (SHE) cells, two preneoplastic cell lines [tumor suppressor stage I (sup +I) and non-tumor suppressor stage II (sup -II)], and hamster tumor cell lines. Stage I preneoplastic cells are nontumorigenic immortal clones that suppress tumorigenicity when hybridized to tumor cells, whereas stage II cells have lost the ability to suppress tumorigenicity in cell hybrids. We refer to these two types of preneoplastic cells as sup +I and sup -II, respectively. Neoplastic progression is generally associated with cellular alterations in growth factor responsiveness. Therefore, to study the regulation of apoptosis in the system described above, cells were cultured in low serum (0.2%) as a means of withdrawing growth factors. In low serum, normal SHE cells were quiescent (labeling index of 0.2%), with little cell death. The sup +I cells showed a relatively low labeling index (1.6%) but, in contrast to the normal cells, died at a high rate (55% cell loss after 48 h) by apoptosis, as evidenced by morphology, DNA fragmentation, and in situ end-labeling of fragmented DNA. The apoptotic cells did not go through a replicative cycle while in low serum, implying that apoptosis was initiated in the G0/G1 phase of the cell cycle. The sup -II cell line showed a high labeling index (40%) after 48 h, but cell growth was balanced by cell death that occurred at approximately the same rate. The cells died, however, predominantly by necrosis. The tumor cell lines continued to proliferate in low serum, with high labeling indices (ranging from 27% to 43%) and a low level of apoptotic or necrotic cell death. To determine the relative ability of these cells to survive in vivo, normal SHE cells, sup +I cells, and sup -II cells were injected s.c. into nude mice. At 5 or 21 days after injection, the normal SHE cells were readily retrieved from the mice and grew well in culture. In contrast, few sup +I cells were retrieved 5 days after injection and no viable cells were retrieved after 21 days. Sup -II cells were not retrieved at either the 5-day or 21-day harvest, and histological examinations of the sites of injection showed the presence of macrophages, eosinophils, and neutrophils, indicating an inflammatory response associated with necrotic cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Isolation and characterization of a 1-beta-D-arabinofuranosylcytosine-resistant Chinese hamster ovary cell mutant that is also X-ray sensitive and is noncomplementary with ataxia telangiectasia cells.

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray-sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair. Such mutant cells could also be used for the isolation and characterization of human DNA repair genes. In particular, it was predicted that the repair gene defective in individuals with ataxia telangiectasia (AT) might be amenable to study with ara-C-resistant (X-ray-sensitive) mutants, since additional studies, presented here, have shown that AT cells are resistant to ara-C. In the long term, it is hoped that determining the specific defect in AT might lead to an understanding of the possible role of defective repair in tumor induction and/or progression. The general approach used to isolate ara-C-resistant Chinese hamster ovary cell mutants was to treat cells with ethyl methanesulfonate and select in increasing concentrations of ara-C. Although several mutants were isolated, one in particular, Ara-CR213, has been studied most extensively. It was selected largely because it shows the greatest sensitivity to X-rays. Ara-CR213 cells were hypersensitive to the killing effect of X-rays with an LD10 of 2.5 Gy as compared to the wild-type cells that had an LD10 of 6 Gy. The mutant showed an increased frequency of X-ray-induced chromosomal aberrations in the G1 and G2 stages of the cell cycle compared to wild-type frequencies. There was no increase in sister chromatid exchange levels. All of these observations in Ara-CR213 are very similar to those made with AT cells in our and other laboratories. Even more important, complementation analysis of Ara-CR213 x AT hybrid cells indicated that the gene responsible for X-ray sensitivity of AT is also mutated in Ara-CR213 cells. Thus, Ara-CR213 appears to have a mutant phenotype and probably genotype that is very similar to, if not exactly the same as, those of AT. This makes it quite different from other X-ray-sensitive cells that have been isolated in other laboratories.

Animals↗
PubMed1987

Modelling the nursing homes system: bed growth, patient turnover and the availability of nursing home places.

This paper uses two simple mathematical models to quantify the potential impact of policy changes on the operation of the nursing home system. The first model shows that the effect of bed growth on admission rates can be substantial and that consequently a slowdown of bed growth can result in a significant decrease in the number of beds becoming available. The second model shows the effect that the balance of high and low turnover patients amongst admissions can have on patient composition and on aggregate admission rates. It is demonstrated that decreasing the proportion of low turnover patients amongst admissions and increasing the turnover of the low turnover group are the most effective ways of increasing the availability of nursing home places. Moreover, such increased availability can be achieved without any increase in bed numbers.

Aged↗
PubMed1986

Analysing access to nursing home care.

The aim of this study is to investigate access to nursing home care in selected regions of Australia, Canada and the United States, and to examine the common ways in which nursing homes are used. Firstly, a review of methodological considerations in measuring access to nursing home care is made. Secondly, patient turnover patterns are interpreted with a view to showing differences in nursing home use among the countries studied; aggregate turnover rates, length of stay and outcomes are compared. Thirdly, groups of patients who differ in demographic and morbidity characteristics and in their use of nursing homes are discussed. Finally a number of distributive implications of these results are raised and a framework is outlined for considering redistributive consequences of changes in the use of nursing homes. It is concluded that the rate of flow of patients through nursing homes is as important a determinant of access to nursing home care as the level of bed provision and that adoption of this dynamic view of access indicates considerable scope for redistributing use of resources within the nursing home systems of all three countries.

Aged↗
PubMed1986

Dementia in elderly adults: prevalence and institutionalization.

A formula for age-specific dementia prevalence is derived using data from a New Zealand study. Six other studies of dementia prevalence selected according to certain criteria are analyzed and their results shown to be not significantly different from those expected on the basis of the formula. Moreover, the small standard error estimate for the studies in aggregate indicates that the observed results are in close agreement with those expected. Dementia prevalence is estimated in both current and projected Australian aged populations using the age-specific prevalence formula. Comparison of the number of confused persons in institutions with the estimated number in the total population suggests that almost half of the moderate or severe dementia cases may be institutionalized, a much higher proportion than reported in other studies. Variations in overall levels of institutionalization are suggested as the main reason for such differences.

Age Factors↗
PubMed1981

The effect of private isolation rooms on patient care practices, Colonization and infection in an intensive care unit.

Conversion of an intensive care unit (ICU) from an open unit to isolation rooms permitted study of patient care practices, colonization and infection in both settings. Air sampling and observation of patient care practices included 99 of 410 open unit patients (168 patient-hours during nine months) and 68 of 1,022 isolation room patients matched on the basis of risk factors for infection and staff contact (113 patient-hours during 12 months). Number and type of interactions between staff and patients, and frequency of handwashing and its relationship to patient-staff interactions were recorded. All ICU patients were monitored daily for signs of and selected risk factors for infection, and material for culture for six surveillance organisms was obtained every four days. Numbers of persons interacting with a patient hour were 6.1 +/- 3.5 in the open units and 4.9 +/- 2.8 in the isolation rooms (0.05 less than P less than 0.10). Frequency of handwashing did not increase significantly in the unit providing convenient sinks, occurring in an observed to expected ratio of only 24 percent. Over-all rates of infection in the open unit and isolation rooms were 15.0 and 13.4, respectively. Half of the infections occurring in patients with complete cultures obtained on admission were caused by organisms colonizing the patient upon admission to the ICU. The isolation rooms did not appear to reduce nosocomial acquisition (P = 0.168, Mantel-Haenszel) of the six surveillance organisms. We conclude that many patient-staff interactions in an ICU are not followed by handwashing, and that the new unit design had no apparent effect upon the frequency of handwashing or over-all incidence of colonization and infection in the ICU.

Air Microbiology↗
PubMed1980

The effect of temperature on times to hatching of eggs of the nematode Ostertagia circumcincta.

A delayed gamma distribution satisfactorily described the distribution of times to hatching of Ostertagia circumcincta eggs incubated in 0.1% saline at temperatures between 6 and 20 degrees C. Below 6 degree C hatching of eggs was extremely variable. The relationship between rates of development and temperature within the range 10 to 20 degrees C was more closely described by a non-linear function than a linear one. The non-linear function was incorporated into a temperature summation equation which satisfactorily predicted the hatching of eggs incubated under conditions of alternating temperatures.

Animals↗