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Biomedical subjects

S Kennedy

Publications and source records attributed to S Kennedy.

At least 37 records · Page 2Linked to original sources
PubMed2003

Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis.

Glarea lozoyensis produces pneumocandin B(0), a potent inhibitor of fungal glucan synthesis. This industrially important filamentous fungus is slow-growing, is very darkly pigmented, and has not been easy to manipulate genetically. Using a PCR strategy to survey the G. lozoyensis genome for polyketide synthase (PKS) genes, we have identified pks1, a gene that consists of five exons interrupted by four introns of 56, 400, 50 and 341 bp. It encodes a 2124-amino acid protein with five catalytic modules: ketosynthase, acyltransferase, two acyl carrier sites, and thioesterase/Claisen cyclase. The transcriptional initiation and termination sites were found 43 bp upstream of the translational start codon and 295 bp downstream of the translational stop codon, respectively. Cluster analysis of 37 fungal ketosynthase modules grouped the Pks1p with PKSs involved in the biosynthesis of 1,8-dihydroxynaphthalene melanin. Disruption of pks1 yielded knockout mutants that displayed an albino phenotype, suggesting that pks1 encodes a tetrahydroxynaphthalene synthase. Gene replacement was achieved by Agrobacterium-mediated transformation, which proved to be simple and efficient. Loss of pigmentation occurred in more than half the transformants, and examination of six non-pigmented transformants showed that the functional genomic copy of the pks1 gene had been replaced by the disruption cassette in each case. A putative 1215-bp ORF (dsg) devoid of introns was present downstream from pks1. BLAST analysis of the 405-amino acid sequence of its predicted product showed a high degree of similarity with Zn(II)(2)Cys(6) binuclear cluster DNA-binding proteins, a class of fungal transcription factors involved in the regulation of polyketide production and other pathways.

Amino Acid Sequence↗
PubMed2003

Lack of prognostic effect of Cox-2 expression in primary breast cancer on short-term follow-up.

AIMS: Cyclo-oxygenase (Cox) catalyses the conversion of arachidonic acid into prostaglandins (PG) and other eciosanoids. The prostaglandins, especially PGE(2) are implicated in tumorigenesis via angiogenesis and suppression of immune reactivity. There are two known isoforms of the enzyme, Cox-1, which is constitutively expressed and the inducible isoform, Cox-2. Cox-2 is induced in response to inflammatory mediators, growth factors, oncogenes and mitogens. Non-selective Cox inhibitors may reduce the relative risk of colonic and breast carcinoma. METHODS: We studied the expression of Cox-2 by immunohistochemistry in 106 primary breast carcinoma specimens collected over a three-year period, using a commercially available polyclonal antibody on formalin-fixed, paraffin-embedded tissue. The slides were examined independently by two pathologists. Tumours were classified according to accepted criteria and an immunohistochemical score (IHS) was calculated for each specimen. The IHS combines the percentage of immunoreactive cells (quantity score) and an estimate of staining intensity (staining intensity score). RESULTS: All patients were female. The mean age was 53 years, range 28-86 years. Forty percent (n=42) of tumours were node negative and 60% (n=64) node positive. Forty-nine percent (n=52) of tumours were grade 3, a further 49% (n=52) grade 2 and 2% (n=2) grade 1. There was no statistically significant correlation between IHS and tumour size, grade, histology, nodal status, estrogen receptor or progesterone receptor positivity. A trend was observed showing an IHS of zero is associated with prolonged survival compared with an IHS of 9-12. CONCLUSION: Cox-2 expression in primary breast cancer does not correlate with accepted pathological or biochemical prognostic indicators.

Adult↗
PubMed2003

Voluntary freewheel running selectively modulates catecholamine content in peripheral tissue and c-Fos expression in the central sympathetic circuit following exposure to uncontrollable stress in rats.

Modulation of sympathetic drive to the spleen is one potential mechanism whereby physical activity prevents stress-induced splenic immune suppression in rats. The current study tested the hypothesis that voluntary freewheel running reduces peripheral sympathetic drive by modulating stress-induced activity of brain regions synaptically linked to sympathetically innervated peripheral organs, including the adrenals and spleen. To this end, adrenal and splenic catecholamine content and activity of the central sympathetic circuit indexed by c-Fos protein induction, elicited by acute exposure to inescapable tail shock, were measured. Stressor exposure depleted adrenal and splenic norepinephrine content and elicited a robust increase in c-Fos in the brains of sedentary rats. Physical activity status had no effect on adrenal norepinephrine content. Indicative of attenuated sympathetic drive to the spleen, however, 6 weeks of voluntary freewheel running diminished stress-induced splenic norepinephrine depletion, and significantly attenuated stress-induced c-Fos in specific brain regions responsible for sympathetic regulation, including tyrosine hydroxylase-immunoreactive neurons of the locus coeruleus, A5 cell group and rostral ventrolateral medulla. Results suggest that voluntary activity attenuates sympathetic drive to the spleen during stressor exposure by selectively modulating stress-induced activity of the central sympathetic circuit. The attenuation of sympathetic responses observed in this study may be one important mechanism for the protective effect of physical activity against stress-related illness and immunosuppression.

Adrenal Glands↗
PubMed2003

Apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with porcine rubulavirus.

In a first experiment, five pigs were inoculated intranasally with porcine rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2(+) and CD8(+) T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4(+) and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2(+), CD8(+) and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

Age Factors↗
PubMed2003

Visceral manifestations in hereditary haemorrhagic telangiectasia type 2.

Hereditary haemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterised by epistaxis, telangiectases, and visceral manifestations. The two known disease types, HHT1 and HHT2, are caused by mutations in the endoglin (ENG) and ALK-1 genes, respectively. A higher frequency of pulmonary arteriovenous malformations (AVMs) has been reported for HHT1 while HHT2 is thought to be associated with a lower penetrance and milder disease manifestations. In this study, we present 10 families with an ALK-1 genotype. Visceral manifestations were detected in 24 (26%) of the 93 HHT2 patients from nine of the families and included gastrointestinal bleeding (14%), intrahepatic shunts (6%), and AVMs in the lung (4%) and brain (3%). Gastrointestinal bleeding, the most frequent visceral manifestation, was reported in six of the 10 families, mostly in patients over the age of 50. These patients also had frequent epistaxis and suffered from anaemia, often requiring blood transfusions. The identification of ALK-1 mutations in subjects with a suspected diagnosis and without clinical signs of HHT argue in favour of a molecular diagnosis. We also analysed the data published on 44 families with HHT2 and conclude that visceral manifestations occur in 26 of these families and affect 30% of HHT2 patients. This is considered an underestimate given incomplete and variable screening for lung, brain, and/or liver involvement in different clinical centres. These findings, however, stress the need for an early diagnosis of HHT that can be useful for the early control of associated visceral involvement.

Activin Receptors, Type I↗
PubMed2002

First report of post-weaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome in pigs in Greece.

Post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) are two recently described conditions of pigs at the late nursery and fattening stages. The aim of this short communication was to describe the first reported occurrence of these conditions and of porcine circovirus type 2 (PCV2) infection in Greece. The clinical signs, gross post-mortem changes and histopathological changes observed in affected pigs, were similar to those previously described for both PDNS and PMWS. As in previous reports, the lesions were associated with PCV2 infection, which was demonstrated by immunohistochemical and in situ hybridization methods.

Animals↗
PubMed2002

A systematic review of the accuracy of ultrasound in the diagnosis of endometriosis.

OBJECTIVE: To evaluate transvaginal and transabdominal ultrasound scanning, with or without Doppler, as a diagnostic test for the accurate diagnosis of pelvic endometriosis. METHODS: The MEDLINE (1966-2001) and EMBASE (1980-2001) databases were searched for relevant studies, published in English. Only studies fulfilling predefined criteria were selected. An assessment of quality was made for each study, and data were then reanalyzed using likelihood ratios to determine the usefulness of the test. RESULTS: In total, 67 papers were identified using the search strategy, of which 17 described relevant studies. Of these, seven fulfilled the inclusion criteria. All seven related to the use of transvaginal gray-scale imaging in the diagnosis of ovarian endometriomata specifically, rather than endometriosis. The positive likelihood ratios ranged from 7.6 to 29.8, and the negative likelihood ratios ranged from 0.1 to 0.4. Confidence intervals were wide. One paper addressed the use of conventional color Doppler with ultrasound: the positive likelihood ratio was 1.2, with a negative likelihood ratio of 0.4. One paper assessed the use of color Doppler energy imaging, and showed a positive likelihood ratio of 33.5 and a negative likelihood ratio of 0.1. CONCLUSIONS: Transvaginal ultrasound appears to be a useful test both to make and to exclude the diagnosis of an ovarian endometrioma.

Endometriosis↗
PubMed2002

The effects of immuno-modulation on the clinical and pathological expression of postweaning multisystemic wasting syndrome.

Post-weaning multisystemic wasting syndrome (PMWS), primarily caused by porcine circovirus type 2 (PCV-2), is an economically important disease of pigs in many countries. A trial was designed to investigate the hypothesis that non-specific immuno-modulation can influence the clinical and pathological expression of PMWS in pigs naturally infected with PCV-2. Eighty-four pigs on a commercial pig farm were allocated to three groups of 28 pigs each, during an outbreak of PMWS. The pigs in the first group received an intramuscular injection of a vaccine against Mycoplasma hyopneumoniae (RespiSure, Pfizer, NY, USA) at each of 7 and 28 days of age, followed by an intramuscular injection of normal saline at 42 days of age. The animals in the second group received, by intramuscular injection, normal saline at 7 days of age followed by a non-specific immuno-modulating drug (Baypamun, Bayer, Leverkusen, Germany) at each of 28 and 42 days of age. The pigs in the third (control) group received an intramuscular injection of normal saline on each of 7, 28 and 42 days of age. The trial was concluded when the pigs had reached the age of 73 days. Clinical signs characteristic of PMWS developed in 42.9% of pigs inoculated with RespiSure and in 50% of pigs treated with Baypamun; six pigs from each of these groups died. Moderate to severe gross and histopathological lesions of PMWS, associated with abundant PCV-2 antigen, were seen in a wide range of tissues of pigs from these groups at the end of the trial. In contrast, only 10.7% of pigs in the control group developed clinical signs and only one died. Mild to moderate lesions and scant PCV-2 antigen were occasionally observed in tissues of control pigs at the end of the trial. This is the first study to demonstrate that non-specific stimulation of the immune system by a vaccine or an immuno-modulator drug can potentiate viral replication and increase the severity of clinical signs during an outbreak of PMWS.

Adjuvants, Immunologic↗
PubMed2001

Validation of a technique to measure leukocyte adhesion to arterial segments: effects of drug treatments.

Adhesion and transmigration of leukocytes into arterial walls occurs after vascular injury and may play a role in the development of atherosclerosis and restenosis. This protocol presents a simple, rapid method for quantifying leukocyte adhesion to artery segments ex vivo. The procedure involves isolating leukocytes from rabbit whole blood and labelling with the gamma-emitting isotope 51Cr. Labelled leukocytes are added to open rings of subclavian artery taken from the same rabbit. After gamma counting, percentage leukocyte adhesion can be calculated with reference to a sample containing a quantity of labelled leukocytes equivalent to that which was added to the artery. Leukocyte adhesion was increased by L-NAME, thrombin and increasing incubation time and decreased by low temperatures. In addition, leukocyte adhesion was found to be increased following a vascular stretch injury performed in vitro. This protocol offers a number of advantages: the rapidity of the leukocyte isolation and labelling; the small quantity of leukocytes required; the ability to use autologous leukocytes; the applicability to whole arteries and arteries injured in vitro or in vivo, allowing the effects of vascular injury on leukocyte adhesion to be studied.

Animals↗
PubMed2001

Novel role for RNA-binding protein CUGBP2 in mammalian RNA editing. CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting with apobec-1 and ACF, the apobec-1 complementation factor.

Mammalian apolipoprotein B (apoB) mRNA editing is mediated by a multicomponent holoenzyme containing apobec-1 and ACF. We have now identified CUGBP2, a 54-kDa RNA-binding protein, as a component of this holoenzyme. CUGBP2 and ACF co-fractionate in bovine liver S-100 extracts, and addition of recombinant apobec-1 leads to assembly of a holoenzyme. Immunodepletion of CUGBP2 co-precipitates ACF, and these proteins co-localize the nucleus of transfected cells, suggesting that CUGBP2 and ACF are bound in vivo. CUGBP2 binds apoB RNA, specifically an AU-rich sequence located immediately upstream of the edited cytidine. ApoB RNA from McA cells, bound to CUGBP2, was more extensively edited than the unbound fraction. However, addition of recombinant CUGBP2 to a reconstituted system demonstrated a dose-dependent inhibition of C to U RNA editing, which was rescued with either apobec-1 or ACF. Antisense CUGBP2 knockout increased endogenous apoB RNA editing, whereas antisense knockout of either apobec-1 or ACF expression eliminated apoB RNA editing, establishing the absolute requirement of these components of the core enzyme. These data suggest that CUGBP2 plays a role in apoB mRNA editing by forming a regulatory complex with the three components of the minimal editing enzyme, apobec-1, ACF, and apoB RNA.

APOBEC-1 Deaminase↗
PubMed2001

Mutagenesis of apobec-1 complementation factor reveals distinct domains that modulate RNA binding, protein-protein interaction with apobec-1, and complementation of C to U RNA-editing activity.

C to U editing of apolipoprotein B (apoB) RNA requires a multicomponent holoenzyme complex in which minimal constituents include apobec-1 and apobec-1 complementation factor (ACF). We have examined the predicted functional domains in ACF in binding apoB RNA, interaction with apobec-1, and complementation of RNA editing. We demonstrate that apoB RNA binding and apobec-1-interacting domains are defined by two partially overlapping regions containing the NH(2)-terminal RNA recognition motifs of ACF. Both apoB RNA binding and apobec-1 interaction are required for editing complementation activity. ACF is a nuclear protein that upon cotransfection with apobec-1 results in nuclear colocalization and redistribution of apobec-1 from the cytoplasm. ACF constructs with deletions or mutations in the putative nuclear localization signal (NLS) still localize in the nucleus of transfected cells but do not colocalize with apobec-1, the latter remaining predominantly cytoplasmic. These observations suggest that the putative NLS motif in ACF is not responsible for its nucleo-cytoplasmic trafficking. By contrast, protein-protein interaction is important for the nuclear import of apobec-1. Taken together, these data suggest that functional complementation of C to U RNA editing by apobec-1 involves the NH(2)-terminal 380 residues of ACF.

APOBEC-1 Deaminase↗
PubMed2001

Myc drives apoptosis in PC12 cells in the absence of Max.

A conditionally active chimeric form of the c-Myc protein fused to the ligand-binding domain of the estrogen receptor (MycER) was expressed in PC12 cells. Induction of Myc activity resulted in a threefold increase in apoptosis after 5 days when cells were maintained in 1% serum. The effect of Myc overexpression was dependent on its DNA-binding domain but not on its heterodimeric binding protein Max, which is absent in PC12 cells. Preincubation of the c-Myc overexpressing cells with either NGF or bFGF, but not EGF, prevented the Myc-mediated increase in apoptosis, although the signaling pathways used by NGF and bFGF to block cell death differed. NGF-mediated rescue was mediated by the phosphatidylinositol 3'-OH (P13) kinase/Akt pathway while rescue by bFGF was not affected by P13 kinase inhibitors. These results show that Myc can induce apoptosis in PC12 cells in a Max-independent manner and that alternate signaling pathways exist to mediate cell survival.

Animals↗