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Biomedical subjects

E Perkins

Publications and source records attributed to E Perkins.

At least 19 recordsLinked to original sources
PubMed2005

The expression and activation of matrix metalloproteinase-1 after subarachnoid haemorrhage in rats.

BACKGROUND: Collagen lattice contraction has been reported as another aspect of the pathogenesis of cerebral vasospasm after subarachnoid haemorrhage (SAH). Recently, some authors have suggested that matrix metalloproteinase-1 (MMP-1) plays an important role in collagen lattice contraction. Therefore, this study aimed to clarify a role of MMP-1 during cerebral vasospasm in a rat SAH model. METHOD: We used a single-SAH model in rats and assessed the basilar arteries (BAs) at 30 minutes and on 2 days after SAH by cross-sectional area measurement and other histological parameters. Immunohistochemistry and Western blot analysis were used to quantify MMP-1 expression and activation. RESULTS: BAs in rats significantly exhibited severe cerebral vasospasm at 30 minutes after SAH and moderate vasospasm on Day 2. The immunohistochemistry and Western blotting performed in BAs of rats demonstrated that both expression and activation in MMP-1 peaked at 30 minutes after SAH and then declined to the control level. CONCLUSIONS: MMP-1 is expressed and activated in a parallel time course to the development of cerebral vasospasm in an experimental model of SAH.

Animals↗
PubMed2004

Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy.

A Raman spectroscopy cell-based biosensor has been proposed for rapid detection of toxic agents, identification of the type of toxin and prediction of the concentration used. This technology allows the monitoring of the biochemical properties of living cells over long periods of time by measuring the Raman spectra of the cells non-invasively, rapidly and without use of labels (Notingher et al. 2004 doi:10.1016/j.bios.2004.04.008). Here we show that this technology can be used to distinguish between changes induced in A549 lung cells by the toxin ricin and the chemical warfare agent sulphur mustard. A multivariate model based on principal component analysis (PCA) and linear discriminant analysis (LDA) was used for the analysis of the Raman spectra of the cells. The leave-one-out cross-validation of the PCA-LDA model showed that the damaged cells can be detected with high sensitivity (98.9%) and high specificity (87.7%). High accuracy in identifying the toxic agent was also found: 88.6% for sulphur mustard and 71.4% for ricin. The prediction errors were observed mostly for the ricin treated cells and the cells exposed to the lower concentration of sulphur mustard, as they induced similar biochemical changes, as indicated by cytotoxicity assays. The concentrations of sulphur mustard used were also identified with high accuracy: 93% for 200 microM and 500 microM, and 100% for 1,000 microM. Thus, biological Raman microspectroscopy and PCA-LDA analysis not only distinguishes between viable and damaged cells, but can also discriminate between toxic challenges based on the cellular biochemical and structural changes induced by these agents and the eventual mode of cell death.

Biological Assay↗
PubMed2004

Transfer and stable transgene expression of a mammalian artificial chromosome into bone marrow-derived human mesenchymal stem cells.

Mammalian artificial chromosomes (ACEs) transferred to autologous adult stem cells (SCs) provide a novel strategy for the ex vivo gene therapy of a variety of clinical indications. Unlike retroviral vectors, ACEs are stably maintained, autonomous, and nonintegrating. In this report we assessed the delivery efficiency of ACEs and evaluated the subsequent differentiation potential of ACE-transfected bone marrow-derived human mesenchymal stem cells (hMSCs). For this, an ACE carrying multiple copies of the red fluorescent protein (RFP) reporter gene was transferred under optimized conditions into hMSCs using standard cationic transfection reagents. RFP expression was detectable in 11% of the cells 4-5 days post-transfection. The RFP-expressing hMSCs were enriched by high-speed flow cytometry and maintained their potential to differentiate along adipogenic or osteogenic lineages. Fluorescent in situ hybridization and fluorescent microscopy demonstrated that the ACEs were stably maintained as single chromosomes and expressed the RFP transgenes in both differentiated cultures. These findings demonstrate the potential utility of ACEs for human adult SC ex vivo gene therapy.

Adipocytes↗
PubMed2003

Opportunistic screening for genital chlamydial infection. I: acceptability of urine testing in primary and secondary healthcare settings.

OBJECTIVES: To determine the acceptability of opportunistic screening for Chlamydia trachomatis in young people in a range of healthcare settings. DESIGN: An opportunistic screening programme (1 September 1999 to 31 August 2000) using urine samples tested by ligase chain reaction (LCR). Data on uptake and testing were collected and in-depth interviews were used for programme evaluation. SETTING: General practice, family planning, genitourinary medicine clinics, adolescent sexual health clinics, termination of pregnancy clinics, and women's services in hospitals (antenatal, colposcopy, gynaecology and infertility clinics) in two health authorities (Wirral and Portsmouth and South East Hampshire). Main participants: Sexually active women aged between 16 and 24 years attending healthcare settings for any reason. MAIN OUTCOME MEASURES: Uptake data: proportion of women accepting a test by area, healthcare setting, and age; overall population coverage achieved in 1 year. Evaluation data: participants' attitudes and views towards opportunistic screening and urine testing. RESULTS: Acceptance of testing by women (16-24 years) was 76% in Portsmouth and 84% in Wirral. Acceptance was lower in younger women (Portsmouth only) and varied by healthcare setting within each site. 50% of the target female population were screened in Portsmouth and 39% in Wirral. Both the opportunistic offer of screening and the method of screening were universally acceptable. Major factors influencing a decision to accept screening were the non-invasive nature of testing and treatment, desire to protect future fertility, and the experimental nature of the screening programme. CONCLUSIONS: An opportunistic model of urine screening for chlamydial infection is a practical, universally acceptable method of screening.

Adolescent↗
PubMed2003

Opportunistic screening for genital chlamydial infection. II: prevalence among healthcare attenders, outcome, and evaluation of positive cases.

OBJECTIVES: To determine the prevalence and treatment outcomes among young women screened opportunistically for genital Chlamydia trachomatis and to evaluate the impact of screening in those participating. DESIGN: An opportunistic screening programme (1 September 1999 to 31 August 2000) using urine samples, tested by ligase chain reaction (LCR). In-depth interviews were used for programme evaluation. SETTING: Screening was offered in two health authorities at general practice, family planning, genitourinary medicine (GUM), adolescent sexual health, termination of pregnancy clinics and women's services in hospitals (antenatal, colposcopy, gynaecology and infertility clinics). Main participants: Sexually active women (16-24 years) attending for any reason. MAIN OUTCOME MEASURES: Screening data: prevalence of infection by age and healthcare setting; proportion of positive patients attending for treatment. Evaluation data: participants' attitudes and views towards screening and follow up. RESULTS: In total, 16 930 women (16-24 years) were screened. Prevalence was higher in younger women (16-20) than those aged 21-24 years and was highly variable at different healthcare settings (range 3.4%-17.6%). Prevalence was approximately 9% in general practice. The role of the project health advisers in managing results and coordinating treatment of positive individuals was essential; the vast majority of all positives were known to be treated. Women felt that screening was beneficial. Improving awareness and education about sexually transmitted infections is required to alleviate negative reactions associated with testing positive for infection. CONCLUSIONS: Prevalence of infection outside GUM clinics is substantial and opportunistic screening using urine samples is an acceptable method of reaching individuals with infection who do not normally present at specialist clinics.

Adolescent↗
PubMed2002

Retrofitting of a satellite repeat DNA-based murine artificial chromosome (ACes) to contain loxP recombination sites.

A satellite DNA-based mammalian artificial chromosome (ACes) was generated and subsequently modified by targeting of a loxP-red fluorescent protein (RFP) expression cassette via homologous recombination into a ribosomal DNA (rDNA)-containing locus. Clones containing correctly targeted ACes were identified by PCR from populations of RFP-expressing cells enriched by FACS sorting and were further characterized by fluorescent in situ hybridization. The targeted ACes maintained its ability to be purified to near homogeneity. Studies are currently underway to further characterize the functionality, carrying capacity, stability and transfectability of this modified ACes.

Animals↗
PubMed2001

Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identified using a cell-based screen in yeast.

Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 or exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone. In separate experiments, when a putative protein regulator of PARP activity, human poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored. Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibit PARP1 and PARP2 activity in yeast cell extracts. Thus, our data reflect the strength of using yeast to identify small molecule inhibitors of therapeutically relevant gene families, including those that are not found in yeast, such as PARP. The resultant inhibitors have two critical uses (a) as leads for drug development and (b) as tools to dissect cellular function.

ATP-Binding Cassette Transporters↗
PubMed2001

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.

Amino Acid Sequence↗
PubMed2000

Selenium causes growth inhibition and apoptosis in human brain tumor cell lines.

We examined the effect of the trace element selenium on human glioma cell lines: T98G, U373MG, and U87MG, in addition to dermal fibroblast cells. Cultures were incubated with sodium selenite, and the following parameters were studied: cell growth, mitochondrial function, and ultrastructure. Cell growth was assayed by counting the number of viable cells after treatment with selenium. Mitochondrial function was analyzed using the MTT (tetrazolium salt reduction) assay. Apoptosis was determined by evaluating nuclear chromatin condensation by electron microscopy. The results indicated that selenium had a significant inhibitory effect on the growth of the tumor cells but had little effect upon dermal fibroblasts which had been passaged numerous times. Selenium also induced mitochondrial damage as shown by MTT assay in two brain tumor cell lines and in minimally passaged fibroblasts, but it had little effect upon the high-passage fibroblasts. Ultrastructurally, mitochondria had electron-dense inclusions resulting from selenium treatment. High rates of apoptosis were induced by selenium in the tumor cell lines and in the minimally passaged fibroblasts, whereas the fibroblasts with a high number of passages had some resistance to selenium treatment. This study correlates the adverse effects of selenium on mitochondrial function, inhibition of cell growth, and apoptosis and shows that selenium similarly affects three different brain tumor cell lines and minimally passaged fibroblasts. Further, the results with fibroblasts show that some types of cells after repeated passages can develop resistance to selenium damage.

Apoptosis↗
PubMed1999

Sequential imaging and volumetric analysis of an intracerebral C6 glioma by means of a clinical MRI system.

In this study, using high resolution coils; implanted growing rat brain tumors were imaged sequentially with 3-D volume measurements generated by means of a clinical magnetic resonance imaging system (CMRI) and commercially available wrist coil. Ten female Sprague-Dawley rats were used, eight were implanted with C6 rat glioma cells and two served as controls. The images that were used for the three-dimensional (3-D) measurements were obtained from T1 weighted post contrast sequences. A commercially available computer work station with 3-D image analysis software was used to generate the tumor volumes. In addition to the rat studies a mouse was included to see if the resolution would be adequate for imaging very small brains. Six rats had brain tumor growth after transplantation and two rats did not have any tumor growth, however, their images were similar to the controls animals. Tumor volumes varied widely among the implanted rats. The number of implanted tumor cells had no direct relationship to developing tumor volumes. This study demonstrates that high resolution images of a rat brain tumor can be obtained from a CMRI system using a commercially available wrist coil which is capable of imaging two rats at the same time or even a mouse brain. A commercially available computer work station was able to generate the tumor volumes. The ability to image brain tumor and generate volume measurements over time has potential for animal research.

Animals↗
PubMed1999

Long-term remission of malignant brain tumors after intracranial infection: a report of four cases.

OBJECTIVE: This report describes four patients with malignant brain tumors in whom regression or cure seems to be related to infection with bacteria. METHODS: An analysis of the four clinical cases reported and a review of the literature produced a comprehensive body of both experimental and clinical data concerning the antineoplastic properties of bacteria. RESULTS: Although direct oncolytic effects from bacteria have been suggested, immune adjuvant responses to tumor suppression are emphasized. In one of our patients, infiltration of numerous granulocytes and lymphocytes into the tumor at the time of initial surgery was observed, suggesting that a spontaneous immune reaction had begun. Also, in two other patients, tumor aggression occurred in association with a bacterial process that was not in direct contact with the tumor. In three of the cases described, Enterobacter aerogenes was recovered from the microbial cultures. Whether the presence of this organism was coincidental or whether this organism plays an important role in tumor defense is not known; however, a specific cross-reactive immunological attack to the tumor is suggested. CONCLUSION: The case histories presented in conjunction with the relevant literature reviewed support the concept that microbial infections may influence immune responses in brain tumor defense.

Adult↗
PubMed1997

A public health approach to nursing in the community.

This article, the first in a three-part series on public health, promotes the case for community nurses to grasp the opportunities a public health approach to care offers to populations and communities. The importance of organisation and evaluation are emphasised as is the need for support from colleagues which, argue the authors, can only be obtained if the benefits of this approach are made clear. The second article in the series will appear next week.

Attitude of Health Personnel↗
PubMed1994

Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks.

Rearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homologous or diverged (19%) repeats of the URA3 genes (from S. cerevisiae or S. carlsbergensis) separated by the genetically detectable ADE2 colour marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.

DNA Damage↗
PubMed1993

Ascorbic acid-enhanced antiproliferative effect of flavonoids on squamous cell carcinoma in vitro.

We examined the effects of flavone and two polyhydroxylated plant flavonoids (quercetin and fisetin), either singly or in combination with ascorbic acid, on the growth of a human squamous cell carcinoma cell line (HTB 43) in vitro. Fisetin and quercetin significantly impaired cell growth in the presence of ascorbic acid. Exposure of cells to ascorbic acid (2 micrograms/ml) and 2 micrograms/ml of either fisetin or quercetin resulted in 61 and 45% inhibition of cell growth, respectively, in 72 h, while treatment with ascorbic acid alone had no effect on cellular proliferation. Flavone and ascorbic acid, either as single agents or in combination, exhibited no significant inhibition at any of the concentrations tested. The enhancement of the antiproliferative effect of the above flavonoids by ascorbic acid may be due to its ability to protect these compounds against oxidative degradation.

Adjuvants, Pharmaceutic↗
PubMed1993

Inverted DNA repeats: a source of eukaryotic genomic instability.

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.

Base Sequence↗
PubMed1993

Cerebrovascular histopathology after intracarotid infusion of Gelfoam in the rat.

To evaluate the histopathology of Gelfoam on the cerebral vasculature, 42 Sprague-Dawley rats weighing 250 to 300 g received internal carotid artery infusions of 0.3 ml of Gelfoam solution (5 mg/ml) or normal saline (0.9%). The animals were killed at 1 hour, 5 hours, 3 days, 1 week, 2 weeks, and 4 weeks after the infusion. The brains were removed, sectioned, and stained with hematoxylin and eosin. Examination of brain sections revealed Gelfoam emboli lodged primarily in the small leptomeningeal arteries. At 5 hours after infusion, inflammatory cells were noted in the arterial walls with vessel occlusion. Further canalization of vessels was noted at 1 week. At 4 weeks, Gelfoam was not seen in the specimens. This study suggests that Gelfoam acts as an embolic agent in vessels over short periods of time with no residual inflammatory activity postinfusion.

Animals↗
PubMed1992

Transposon Tn5 excision in yeast: influence of DNA polymerases alpha, delta, and epsilon and repair genes.

Interaction between short repeats may be a source of genomic rearrangements and deletions. We investigated possible interactions between short (9 base pairs) direct repeats in yeast by using our previously described system for analyzing bacterial transposon Tn5 excision in yeast. Mutations of either POL3 or POL1, the proposed structural genes for polymerases delta and alpha, respectively, yield high levels of excision at semipermissive temperatures. pol2 (corresponding to polymerase epsilon) and pol2 pol3 double mutants do not exhibit enhanced excision. A majority of excision events involve direct repeats and are precise; the remaining imprecise excisions occur within or in the vicinity of the repeats. The three DNA repair pathways identified by rad1, rad6 and rad18, rad50 and rad52 mutations were examined for their possible role in Tn5 excision; no enhancement was observed in mutants. However, the pol3-stimulated Tn5 excision was reduced in rad52 and rad50 mutants. This suggests the potential for interaction between the systems for DNA double-strand break/recombinational repair and DNA synthesis. Based on the suggestion of Morrison et al. [Morrison, A., Araki, H., Clark, A. B., Hamatake, R. H. & Sugino, A. (1990) Cell 62, 1143-1151] that polymerases delta and alpha are responsible for lagging-strand synthesis and that polymerase epsilon is responsible for leading-strand synthesis, we suggest that Tn5 excision is stimulated under conditions of altered lagging-strand synthesis, possibly due to extended opportunities for single-strand interactions between the inverted insertion sequence I550 repeats of Tn5.

Base Sequence↗