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Cell wall-deficiency in Staphylococcus aureus and its role in antibiotic resistance

Tue, 01 Jan 2008 00:00:00 GMT

Title: Cell wall-deficiency in Staphylococcus aureus and its role in antibiotic resistance Authors: Fuller, Elizabeth R Abstract: Cell Wall-Deficiency in Staphylococcus aureus and its Role in Antibiotic Resistance. Elizabeth R. Fuller. Cell wall-deficient bacteria (CWDB) induced from Staphylococcus aureus ATCC 9144 (Oxford strain) were generated on medium with elevated osmolality in the presence of sublethal levels of penicillin G. On removal of antibiotic pressure the cell wall-competent (CWC) revertants along with these CWDB exhibited high-level penicillin and methicillin resistance, which was stable in the revertants. The revertants looked visually different, had an altered Gram stain and growth rate. Their matrixassisted laser desorption/ionisation time-of-flight (MALDI-TOF) `fingerprint' was also altered and they were more resistant to lysis by lysostaphin in comparison to the wild-type. Reversed-phaseh igh-performance liquid chromatography( RP-HPLC) showed that the revertants' cell walls had shorter glycan chains and more pentaglycine cross-bridges. A rapid,r eproduciblem ethodu sing liquid mediaw ase stablishedu singt he same medium and sublethal levels of penicillin G. The revertants produced using this method had the same characteristics as those cells produced from the original method. The high-level resistance seen in the revertants was homogenous and confirmed to be due to the transient CWD state, along with not being strain-specific. Transmission electron microscopy showed that the CWD cells and the revertant cells, when grown in penicillin, had a very disordered cell wall with areas where the cell wall appeared absent and were indistinguishable. The revertant cells were mecA-negative,ß -lactamase-negativea nd did not contain any mutations in the coding regions of pbp genes. The CWD cells and revertant cells, when grown in penicillin, were resistant to lysis by lysostaphin but were very sensitive to lysis with Triton X- 100. These data indicate that the resistant cells are not dependent upon an intact cell wall for osmotic stability and they are able to switch readily to this mode of growth in the presence of penicillin G. Description: MD

Studies on whole-body nitrogen turnover, protein synthesis and breakdown in man using 15N glycine

Sat, 01 Jan 1994 00:00:00 GMT

Title: Studies on whole-body nitrogen turnover, protein synthesis and breakdown in man using 15N glycine Authors: Clarke, David Abstract: The experimental work described in this thesis was conducted in the surgical research laboratories of Dr Francis D. Moore in the Peter Bent Brigham Hospital (now the Brigham & Women's Hospital) Boston, Massachusetts, USA, between 1978-1979. It formed part of an ongoing programme of research into protein metabolism in man; specifically to measure total body nitrogen turnover and hence protein synthesis and breakdown, initially in normal volunteers receiving various intravenous feeding regimens, and subsequently in patients. The previous year, 1977, had seen the publication of 'Substrate Interaction in Intravenous Feeding' by Bruce Wolfe et al., from the same laboratories. This was an extensive piece of work incorporating many studies and compared nitrogen balance data together with biochemical, hormonal and plasma amino acid data in normal men fed intravenously with a variety of regimens. Shortly afterwards a series of protein turnover studies was embarked upon, using the uN glycine method, and in collaboration with Dr Vernon Young of the Massachusetts Institute of Technology. The first experiments were essentially a repeat of the studies described by Wolfe et al. (vide supra) but in addition nitrogen turnover, protein synthesis and breakdown were estimated using a continuous 60 hour infusion of uN glycine and measuring enrichment of urinary urea with uN when a plateau was reached. Normal volunteers were studied firstly on normal oral diet and then on a iv succession of intravenous regimens:- amino acids alone (AA), amino acids plus 'high dose' glucose (AA + HOG), amino acids plus fat emulsion (AA + FE), amino acids plus 'low dose' glucose (AA + LOG), amino acids, fat emulsion and low dose glucose (AA + LDG+ FE), and finally 'low dose' glucose alone (LOG). The studies on normal diet, AA and AA+HOG were conducted by Andrew Sim (a Glasgow/Harvard exchange fellow) and Bruce Wolfe. The author took no practical role in these experiments, but was responsible for analysis of the data and the protein metabolism calculations, and was a co-author when the work was published in 1979 (Sim et al., Glucose Promotes Whole-Body Protein Synthesis from Infused Aminoacids in Fasting Man, Lancet i, 68-71). Subsequently, the author did the experiments using AA + LOG + FE, AA + FE, and AA + LOG and LOG. The results on these four regimens were incorporated in a paper presented in 1979 at the Tripartite Meeting of the Surgical Research Society at Oxford under the title 'Isotope Studies of substrate interaction in parenteral nutrition', and also at the 2nd European Congress on Parenteral and Enteral Nutrition at Newcastle upon Tyne in 1980, and later published as 'The Effect of Fat Infusion on Protein Metabolism' (Acta. Chir. Scand., Suppl. 507, 475-484, 1981). When the studies on the various intravenous feeding regimens were completed, attention was turned to the possible distorting effects of variables such as exercise and diet v on the behaviour of the isotope equilibrium curve and plateau. Such effects, if present, might have significance when studies were carried out on patients at a later stage in the research programme. Because each study lasted 48-60 hours, changes might occur either unintentionally or as a result of the needs of clinical management, and if they affected the plateau would alter the resultant calculations of turnover, synthesis and breakdown. Such a potential source of error clearly needed investigation. A pilot study was done in two subjects, later repeated on each, to observe any effects on the curve and plateau of both doubling protein intake and bicycle exercise. Subsequently, more extensive studies were done varying the protein and energy intakes, both orally and intravenously, allowing a detailed analysis of curve perturbation, and introducing the concept of basal catabolic rate. Finally, protein turnover, synthesis and breakdown were estimated seven times in four seriously ill patients. All of the studies mentioned above form the basis of the thesis. Unfortunately, the gestation period of this thesis has been long. There are two main reasons for this. Firstly, the work done was part of a five-year programme of research, with the intention of publishing a paper in a scientific journal at the completion of each stage. This was done vi with the first three regimens (normal diet, AA and AA + HDG) but not with the last four (AA + FE, AA + LOO+ FE, AA + LOG, LDG), although the results were presented at two scientific meetings. Shortly after returning to the United Kingdom the author was appointed a consultant surgeon and this career move assumed priority. Secondly, although it was intended to publish the perturbation studies, it proved impossible to reduce the size of the text to a manageable level suitable for publication in the form of a scientific paper. However, despite the long interval since the experiments were done, no similar work has been published. In particular, virtually no attention has been paid to intentional perturbation. Also, whereas there was a spate of interest in protein turnover studies in the late 1970s and early 1980s, virtually no publications have appeared since 1985. It seems that the potential applications of the method are considered exhausted, and interest has been lost, rather as it was in the 1950s following a short flurry of activity exploring the first cumbersome technique. Hence, it seemed all the more pertinent, even at this late stage, to publish the work in the form of a thesis which could describe in chronological order the continuum of studies as briefly mentioned above. In order to preserve such a progression, the following Introduction contains, with few exceptions, only references up to the time that the experimental studies were commenced, 1978, but the subsequent Discussion(s) in the various sections will attempt to include the relevant literature up to the present time. Description: M.D.

Functional analysis of TSPY and its role in prostate carcinogenesis

Sat, 01 Jan 2005 00:00:00 GMT

Title: Functional analysis of TSPY and its role in prostate carcinogenesis Authors: Omar, Mahmoud Mustafa Abstract: Testis specific protein Y chromosome encoded (TSPY) is a multicopy gene located on the human Y chromosome. It has been reported that the human genome harbours between 20 to 40 copies of the gene. Mammalian homologues of human TSPY were also found to be repetitive. The gene is expressed in human foetal and adult testis. The precise function of the expressed TSPY gene product is not fully defined, but it has been postulated to regulate proliferation of testicular spermatogonia. Up-regulation of TSPY expression has been detected in gonadoblastoma, testicular cancer and prostate cancer. The contribution of abnormal expression of TSPY to prostate carcinogenesis has not been investigated. Prostate cancer (CaP) and benign prostate hyperplasia (BPH) are the most common diseases of the human prostate. Prostate cancer is a disease of the elderly and is the second most common cancer and second most common cause of death from cancer among men in UK. In this thesis, the role of TSPY in prostate carcinogenesis was studied in four related aspects. First, TSPY protein and transcript expression pattern in CaP compared to Benign Prostate Hyperplasia (BPH) was studied using a combination of immunohistochemistry and mRNA in situ hybridisation. Second, the ability of TSPY to regulate cellular proliferation was investigated by transfection experiments of the prostate cancer LNCaP cell line. Third, TSPY genomic copy number in prostate cancer was characterised and compared to BPH. Finally, the downstream genes regulated by TSPY were investigated using high density microarray gene profiling method. An anti-human TSPY polyclonal antibody was developed for immunodetection of TSPY expression level in resected prostate tissues. In total, 72 cases of patients with prostate cancer and 20 cases of patients affected with BPH were studied by immunohistochemistry. TSPY was predominantly detected in the prostatic epithelium. In the benign gland, TSPY expression was limited to the basal cells compartment. TSPY expression was significantly up-regulated in prostate cancer when compared to BPH (P<0.0001). Furthermore, increased TSPY expression level was associated with aggressive disease (tumour with high Gleason score; P<0.02) and the presence of bone metastasis at the time of diagnosis (P<0.028). To address the functional role of 3 TSPY in prostate cancer, FLAG-TSPY was cloned and stably transfected into LNCaP cells. The presence of transfected TSPY increased LNCaP proliferation by two fold compared to empty vector control, consistent with a mitogenic function in CaP (P<0.0001). An absolute quantitative real time PCR based on Taqman assay was established and validated. TSPY genomic copy number was determined from comparing 161 samples: CaP-serum (n=47), resected tumour (n=31); BPH-serum (n=27), resected prostate (n=13) and control-serum (n=45). Of the clinical samples analysed, interpersonal variability of TSPY copy numbers was observed with the majority of cases containing between 20 to 50 TSPY copies per genome. Although, there were variability in TSPY copy numbers among individuals, there was no statistically significant correlation between TSPY copy number (serum and prostatic tissue) and the development of prostate cancer. Studying LNCaP stably transfected with TSPY and empty vector control, the key genes mediating the functional effect of TSPY were identified using Affymetrix oligonucleotide microarray method. In total, 332 genes have been altered 1.5 to 90 fold due to the effect of TSPY over-expression. Ten genes were selected and the gene expression levels were confirmed using semi-quantitative RT-PCR method. Gene clustering analysis has indicated changes in genes that regulate cellular differentiation (NRDG1, NF2, C-MAF and BMP11), apoptotic gene (BAX), cell cycle gene (cyclin G), detoxification gene (GST-2A) and genes linked to adhesion (PCDH7a, PLOD2 and IRS 1). In summary, TSPY is over-expressed in prostate cancer. This abnormal expression is linked to prostate cancer progression and metastasis. The up-regulation of TSPY expression is unlikely to be due to increased copy number. Expression of exogenous TSPY in prostate cancer cells enhanced cellular proliferation. The gene meditates its effect by down-regulating the expression pattern of selected differentiation, apoptosis and adhesion genes. Hence, over-expression of TSPY contributes to prostate carcinogenesis. Description: PhD Thesis

The role of interleukin-17 and RANKL in the regulation of bone destruction in arthritis

Thu, 01 Jan 2004 00:00:00 GMT

Title: The role of interleukin-17 and RANKL in the regulation of bone destruction in arthritis Authors: Abusrer, Salma Abstract: Arthritis is a common disease characterised by chronic inflammation as well as bone and cartilage destruction. Proinflammatory cytokines such as interieukin-17( IL-17), aT cell derived cytokine, and Oncostatin M (OSM) a microphage cytokine, are elevated in rheumatoid arthritis (RA). The aim of this study was to characterise the effects of IL-17 and OSM on the expression of receptor activator of NF-kappaBl and (RANKL) and its decoy receptoro steoprotegeri(nO PG)by mesenchymalil neage cells in vitro, in particular synovial fibroblasts and the human osteo sarcoma cell line (MG-63 and SaOS-2) that are potentially involved in this process as they regulate osteoclastogenesis. In addition, the ability of IL-17 to support osteoclastogenesinis vitro was assessed. Human synovial fibroblasts( SFB)from RA and OA patients were contrasted with cell lines MG-63 and SaOS-2 and were treated with IL-17 and/or OSM for up to 48 hr. The expression and production of RANKL and OPG over a time course was assessed in these cells. To investigate osteoclastogenesis peripheral blood mononuclea cells (PBMCs)were cultured with IL-17 either alone or with Macrophage colony stimulating factor (M-CSF). Osteoclastogenesis was analyzed after 21 days by tartrate-resistant acid phosphatase (TRAP),positive multinucleated cell counts, and resorption of ivory slices. This study demonstrated a direct role for IL- 17 in bone and cartilage catabolism through the induction of RANKL and OPG mRNA levels at 6 hr and 48 hr in SFB RA and OA, MG-63 and SaOS-2. Furthermore the combination of IL-17 and OSM showed that the expression of RANKL and OPGw as down-regulated. Co-incubation of IL-17 with or without M-CSF significantly enhanced TRAP+ve multinucleated cell formation. Furthermore cultures of PBMC with IL-17 on ivory slices showed significantly increased resorption and when cocultured with synovial fibroblasts from RA and OA patients further significant resorption. The addition of OPG to IL-17 cultures significantly inhibited the formation of resorption lacunae. The effect of IL-17 on bone resorption in vitro is mostly RANKL-dependent. This finding may lead to the conclusion that IL-17 significantly induces osteoclasto genesis in vitro. Therefore,I L-17 represents a key cytokine involved in the exacerbation of inflammatory joint disease and is an important target for anti-cytokine therapies. Also these results provide proof of the concept that OPG production can result in effective therapies. Description: PhD Thesis