Biomedical subjects

M P Ryan

Publications and source records attributed to M P Ryan.

At least 19 recordsLinked to original sources

PubMed2005

Ralstonia pickettii: a persistent gram-negative nosocomial infectious organism.

Non-fermenting Gram-negative bacilli create a significant problem in clinical settings, being a widespread cause of nosocomial infections. They are opportunistic pathogens that take advantage of underlying conditions and diseases. Ralstonia pickettii, a non-fermenting Gram-negative bacillus, is regarded as being of minor clinical significance; however, many instances of infections with this organism are reported in the literature. Infections can include bacteraemia/septicaemia caused by contaminated solutions, e.g. distilled water, water for injection and aqueous chlorhexidine solutions. Cases of pseudobacteraemia have been recorded in association with R. pickettii, as have many cases of unusual infections, some of which were very invasive and severe, e.g. meningitis, septic arthritis and osteomyelitis. Six cases of death in four separate instances have also been recorded related to R. pickettii. This review illustrates that R. pickettii is a more important pathogen than was thought previously.

Cross Infection↗

PubMed2003

Do resuscitation attempts in children who die, cause injury?

OBJECTIVE: To determine the incidence, type, and pattern of injury related to resuscitation attempts in children who die. DESIGN: Retrospective review of ambulance, hospital, and necropsy case records. METHOD: All children who died aged 0-14 years between 1994 and 1996, and underwent a full necropsy at the Victorian Institute of Forensic Medicine (Melbourne, Australia) were identified. Children who were subject to recognised trauma before resuscitation or died because of a congenital abnormality were excluded. The records of all remaining children were reviewed. Children were grouped according to whether resuscitation was attempted or not. RESULTS: From a total of 346 children who died, 204 (58.6%) were identified as meeting the inclusion criteria. Resuscitation was performed in 153 (75%) children and was started before ambulance arrival in 123 (60.3%) children. Injuries were detected at necropsy in 65 (42.5%) of children who had resuscitation compared with six (11.7%) of children who had no resuscitation (p<0.0001) chi(2) test. All but two of these injuries were of a minor nature consisting principally of bruises or abrasions. Two significant injuries were identified both occurring as a result of readily identifiable resuscitation procedures. The likelihood of injury increased with the length of resuscitation. In children resuscitated for less than 60 minutes the incidence of injury was 27% compared with 62% for children resuscitated for longer ( p<0.0001). CONCLUSION: This study has shown that cardiopulmonary resuscitation commonly causes minor injuries such as superficial bruises and abrasions and the likelihood of such injury increases with the duration of the cardiopulmonary resuscitation. This information should reassure parents and caregivers that basic life support may be instituted without fear of causing significant injury or adversely affecting outcome in the child with cardiorespiratory arrest. Caution must be exercised when attributing significant injuries to resuscitation attempts and alternative causes must be fully investigated.

Adolescent↗

PubMed2001

Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes.

Toward the goal of identifying complete sets of transcription factor (TF)-binding sites in the genomes of several gamma proteobacteria, and hence describing their transcription regulatory networks, we present a phylogenetic footprinting method for identifying these sites. Probable transcription regulatory sites upstream of Escherichia coli genes were identified by cross-species comparison using an extended Gibbs sampling algorithm. Close examination of a study set of 184 genes with documented transcription regulatory sites revealed that when orthologous data were available from at least two other gamma proteobacterial species, 81% of our predictions corresponded with the documented sites, and 67% corresponded when data from only one other species were available. That the remaining predictions included bona fide TF-binding sites was proven by affinity purification of a putative transcription factor (YijC) bound to such a site upstream of the fabA gene. Predicted regulatory sites for 2097 E.coli genes are available at http://www.wadsworth.org/resnres/bioinfo/.

Base Sequence↗

PubMed2001

Human fibulin-4: analysis of its biosynthetic processing and mRNA expression in normal and tumour tissues.

Here, we report the identification of a human orthologue of fibulin-4, along with analysis of its biosynthetic processing and mRNA expression levels in normal and tumour tissues. Comparative sequence analysis of fibulin-4 cDNAs revealed apparent polymorphisms in the signal sequence that could account for previously reported inefficient secretion in fibulin-4 transfectants. In vitro translation of fibulin-4 mRNA revealed the presence of full-length and truncated polypeptides, the latter apparently generated from an alternative translation initiation site. Since this polypeptide failed to incorporate into endoplasmic reticulum membrane preparations, it was concluded that it lacked a signal sequence and thus could represent an intracellular form of fibulin-4. Using fluorescence in situ hybridisation analysis, the human fibulin-4 gene was localised to chromosome 11q13, this region being syntenic to portions of mouse chromosomes 7 and 19. Considering the fact that translocations, amplifications and other rearrangements of the 11q13 region are associated with a variety of human cancers, the expression of human fibulin-4 was evaluated in a series of colon tumours. Reverse transcription-polymerase chain reaction analysis of RNA from paired human colon tumour and adjacent normal tissue biopsies showed that a significant proportion of tumours had approximately 2-7-fold increases in the level of fibulin-4 mRNA expression. Taken together, results reported here suggest that an intracellular form of fibulin-4 protein may exist and that dysregulated expression of the fibulin-4 gene is associated with human colon tumourigenesis.

Amino Acid Sequence↗

PubMed2001

Heterologous expression of lacticin 3147 in Enterococcus faecalis: comparison of biological activity with cytolysin.

Lacticin 3147 is a broad-spectrum, two-component, lanthionine-containing bacteriocin produced by Lactococcus lactis DPC3147 which has widespread food and biomedical applications as a natural antimicrobial. Other two-component lantibiotics described to date include cytolysin and staphylococcin C55. Interestingly, cytolysin, produced by Enterococcus faecalis, has an associated haemolytic activity. The objective of this study was to compare the biological activity of lacticin 3147 with cytolysin. The lacticin 3147-encoding determinants were heterologously expressed in Ent. faecalis FA2-2, a plasmid-free strain, to generate Ent. faecalis pOM02, thereby facilitating a direct comparison with Ent. faecalis FA2-2.pAD1, a cytolysin producer. Both heterologously expressed lacticin 3147 and cytolysin exhibited a broad spectrum of activity against bacterial targets. Furthermore, enterococci expressing active lacticin 3147 did not exhibit a haemolytic activity against equine blood cells. The results thus indicate that the lacticin 3147 biosynthetic machinery can be heterologously expressed in an enterococcal background resulting in the production of the bacteriocin with no detectable haemolytic activity.

Amino Acid Sequence↗

PubMed2001

Strategy for manipulation of cheese flora using combinations of lacticin 3147-producing and -resistant cultures.

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10(7) CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.

Bacterial Proteins↗

PubMed2000

Artificially recruited TATA-binding protein fails to remodel chromatin and does not activate three promoters that require chromatin remodeling.

Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing the HIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at the CHA1 promoter. Finally, we show that activation of the GAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.

Chromatin↗

PubMed1999

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147.

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.

Alanine↗

PubMed1999

Cyclosporine A-induced alterations in magnesium homeostasis in the rat.

The immunosuppressive drug cyclosporine A (CsA) exhibits significant nephrotoxicity. Disturbance of magnesium (Mg) homeostasis may be an important component of this nephrotoxicity. It has been suggested that transmigration of Mg from plasma to tissues may be an important component of CsA-induced alterations in Mg homeostasis. In this study, CsA nephrotoxicity in male Wistar rats was investigated and alterations in Mg homeostasis along with other indices of toxicity were assessed. Animals were dosed daily for 14 days i.p. with CsA (20 mg/kg body weight). Control animals received vehicle alone. CsA toxicity was evidenced by i) lower gain in body weight, ii) reduced thymus/body weight ratio, iii) increased blood urea nitrogen and creatinine, iv) a tendency for reduced plasma magnesium and v) increased urinary Mg excretion and greatly increased fractional excretion of Mg. Tissue Mg analysis did not reveal any changes in thymus or skeletal muscle Mg while Mg in kidney tissue tended to be reduced. Electron microscopy revealed some damage in renal tubules of rats treated with cyclosporine including translucent cytoplasm, vacuolization, rounded and swollen mitochondria, damage to brush border and disruption of basal infoldings. These results indicate that direct renal tubular damage may result from CsA exposure. No evidence was found for CsA-induced movement of Mg from plasma to tissues. CsA-induced altered renal handling of Mg and this renal Mg wasting may be an important consequence of the nephrotoxicity.

Animals↗

PubMed1999

Assays for nucleosome positioning in yeast.

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ATP-Binding Cassette Transporters↗

PubMed1999

Effects of interferon alpha-2b on barrier function and junctional complexes of renal proximal tubular LLC-PK1 cells.

BACKGROUND: Interferon alpha-2b (IFNalpha) treatment of diseases can be accompanied by impaired renal function and capillary leak syndrome. To explore potential mechanisms of IFNalpha-induced renal dysfunction, an in vitro cell culture model system was established to investigate the effects of IFNalpha on barrier function and junctional complexes. METHODS: LLC-PK1 cells were cultured on microporous membranes. Transepithelial resistance (TER) was measured, and the dose- and time-dependent effects of IFNalpha were assessed. The expression patterns of junctional proteins were examined by Western blot analysis and by confocal immunofluorescence microscopy. RESULTS: IFNalpha produced a dose- and time-dependent decrease in TER. The effect was reversible on removal of IFNalpha at doses up to 5 x 103 U/ml. Tyrphostin, an inhibitor of phosphotyrosine kinases, ameliorated the IFNalpha-induced decrease in TER. Increased expression of occludin and E-cadherin was detected by Western blot analysis after IFNalpha treatment. Immunofluorescence confocal microscopy revealed a broader staining of occludin and E-cadherin following IFNalpha treatment, with prominent staining at the basal cell pole in addition to localization at the junctional region. A marked increase in phosphotyrosine staining along the apico-lateral cell border was detected after IFNalpha treatment. CONCLUSIONS: These findings provide evidence that IFNalpha can directly affect barrier function in renal epithelial cells. The mechanisms involve enhanced tyrosine phosphorylation and overexpression and possibly displacement or missorting of the junctional proteins occludin and E-cadherin.

Animals↗

PubMed1999

Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells.

BACKGROUND: Both interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic cytokines that have been implicated in the development of glomerular and tubular injury in various forms of immune-mediated renal disease, including glomerulonephritis. Although TNF-alpha has been shown to stimulate IL-6 production in renal cells in culture, the signaling mechanisms that regulate IL-6 production are not fully understood. The aim of this study was to examine the role of the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathways in regulating TNF-alpha-mediated IL-6 production from both primary human mesangial cells (HMCs) and human proximal tubular (HPT) cells. METHODS: Primary mesangial and proximal tubular cells were prepared from nephrectomized human kidney tissue. Cells were treated for 24 hours with TNF-alpha in the presence and absence of the specific p38 and ERK1,2 MAPK inhibitors SB203580 and PD98059, respectively, either alone or in combination. IL-6 levels in the cell culture media were measured by enzyme-linked immunosorbent assay. MAPK activation was demonstrated by immunoblot for the active kinase (tyrosine/threonine phosphorylated) in whole cell extracts using phospho-specific antibodies. p38 MAPK activity in HPT cells was measured using an in vitro immunokinase assay using ATF2 as the substrate. RESULTS: TNF-alpha (0.1 to 100 ng/ml) stimulated a dose-dependent increase in IL-6 production in both renal cell types. The activation of the p38 and the ERK1,2 MAPKs occurred following TNF-alpha stimulation. The role of these activations in IL-6 production was confirmed by the ability of both inhibitors SB203580 (1 to 30 microM) and PD98059 (0.01 to 10 microM) to inhibit basal and TNF-alpha-stimulated IL-6 production in both cell types. The addition of both inhibitors in combination caused greater decreases in IL-6 production compared with either inhibitor alone. Pretreatment with SB203580 (10 microM) had no effect on basal or TNF-alpha-stimulated phosphorylation of p38 MAPK but completely abolished TNF-alpha-stimulated p38 MAPK activity. PD98059 decreased both basal and TNF-alpha-stimulated phosphorylation of ERK1,2. CONCLUSIONS: This study provides evidence that both the p38 and ERK MAPK pathways are important for the regulation of the production of IL-6 from the proximal tubular and glomerular mesangial regions of the nephron. In response to TNF-alpha, the activation of both pathways leads to IL-6 production. These findings could aid in an understanding of the cellular mechanisms that regulate IL-6 production and could provide insights into possible pharmacological strategies in inflammatory renal disease.

Cells, Cultured↗

PubMed1999

Factors produced by activated leukocytes alter renal epithelial cell differentiation.

The development of tubulointerstitial fibrosis in inflammatory renal diseases has been linked to disease progression to end-stage renal failure. Understanding the interactions of the factors influencing inflammation and activating the fibrotic process, that is, the inflammatory infiltrate and the resident proximal tubular cells, may lead to a determination of the mechanisms that regulate tubulointerstitial fibrosis. We used an in vitro model of human proximal tubule cells that were stimulated with supernatant from activated peripheral blood mononuclear cells (leukocytes) to study the alterations in cellular phenotype, and examined the signaling pathways mediating epithelial-fibroblast like transdifferentiation. Our hypothesis of the proposed sequence of events leading to tubulointerstitial fibrosis is explained.

Cell Differentiation↗

PubMed1999

Cyclosporine A-induced cell cycle arrest and cell death in renal epithelial cells.

We studied the effects of cyclosporine A (CsA) on the proliferation of LLC-PK1 proximal tubule epithelial cells. DNA damage was found to be an early event in CsA nephrotoxicity and could be a sensitive indicator of CsA injury in renal epithelial cells. Cell cycle arrest induced by CsA was coincident with elevated p53 levels. It is possible that trans-activating p21 may mediate the halting of the cell cycle through the CsA-induced accumulation of p53.

Animals↗

PubMed1999

Effect of duration of dominance of the ovulatory follicle on onset of estrus and fertility in heifers.

In cattle, prolonged progestogen treatments following luteolysis result in persistent dominant follicles (DF) that are associated with precise onset of estrus but marked reductions in pregnancy rate (PR). The aim was to determine whether increasing duration of dominance of the ovulatory follicle in heifers affected 1) precision of onset of estrus and 2) the timing and nature of the decline in PR. In Exp. 1, duration of dominance of the ovulatory follicle was controlled by causing corpus luteum (CL) regression at emergence of the second follicle wave (mean duration of dominance of 2.1+/-.3 d, Dm2, n = 11) or first day of dominance of the second DF of the cycle; the latter was combined with insertion of a 3-mg norgestomet ear implant for 2 to 10 d to maintain the second DF for 4 (Dm4, n = 32), 6 (Dm6, n = 19), 8 (Dm8, n = 49), 10 (Dm10, n = 28), or 12 d (Dm12, n = 20). Heifers detected in estrus were inseminated approximately 12 h later with frozen-thawed semen. Durations of dominance of the ovulatory follicle of up to 8 d did not affect (P>.05) PR (Dm2 8/9, Dm4 19/28, Dm6 14/18, and Dm8 34/48 heifers pregnant), but PR in Dm10 heifers (12/23 heifers pregnant) was reduced (P = .05) compared with Dm2 heifers; PR in Dm12 heifers (2/17 pregnant) was less compared with all other treatments (P<.01). Fitting a logistic regression model to the pooled PR data to examine the trend in PR showed that extending the duration of dominance from 2 to 9 d and from 10 to 12 d resulted in a predicted decline in PR of 10 to 25% and a further decline of 35 to 75%, respectively. Onset of estrus was delayed in heifers assigned to Dm4 treatment relative to all other treatments (P<.001); it was less variable than that for heifers on Dm6, Dm8, and Dm10 treatments (P<.1). In Exp. 2, heifers received a PGF2alpha analogue and a norgestomet implant on d 12 of the cycle for 3 or 7 d to give approximate durations of dominance of the preovulatory follicle of 2 to 4 d (Dm2-4, n = 29) or 6 to 8 d (Dm6-8, n = 24), respectively. The PR did not differ (P>.05) between heifers on Dm2-4 (22/29) and Dm6-8 (15/24) treatments, but the interval to onset of estrus was delayed (P<.05) by 7 h in the Dm2-4 heifers. In conclusion, restricting the duration of dominance of the preovulatory follicle to < or =4 d at estrus, results in a precise onset of estrus and a high PR following a single AI at a detected estrus.

Animals↗

PubMed1999

The natural food grade inhibitor, lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows.

Lacticin 3147 is a broad-spectrum bacteriocin produced by the food-grade organism Lactococcus lactis. Lacticin 3147 is active at a neutral pH and has been shown to be bactericidal to streptococci and staphylococci in vitro. The effectiveness of an intramammary teat seal formulation, and a teat seal containing lacticin 3147 was evaluated at drying off in 68 uninfected quarters of 18 cows. Following infusion of either teat seal or lacticin 3147 combined with teat seal, a deliberate infection challenge of Streptococcus dysgalactiae (approximately equal to 1.5 x 10(4) cfu per teat) was administered by direct inoculation into the teat sinus. During an 8-d experimental period following inoculation, 61% of control quarters and 6% of the treatment quarters either developed clinical mastitis or were shedding the challenge organism. Randomly amplified polymorphic DNA polymerase chain reaction genetic typing was used to confirm that both the new infections and the bacteria surviving in the teats at the end of the experiment were the challenge strain. The combination of teat seal and lacticin 3147 was well tolerated within the udder and elicited only a temporary increase in somatic cell count to 5.7 x 10(5)/ml (88 h after infusion) in a previously uninfected lactating udder quarter. Therefore, we concluded that this nonantibiotic approach to mastitis prevention may contribute to a reduction in the routine application of antibiotics at drying off in the future.

Animals↗

PubMed1999

The natural food grade inhibitor, lacticin 3147, reduced the incidence of mastitis after experimental challenge with Streptococcus dysgalactiae in nonlactating dairy cows.

Animals↗

PubMed1998

Changes in regional renal blood flow after unilateral nephrectomy using the techniques of autoradiography and microautoradiography.

PURPOSE: To determine alterations in regional renal blood flow following unilateral nephrectomy using an autoradiographic technique. The role of prostaglandins and the sympathetic nervous system in the mediation of these changes was assessed. MATERIALS AND METHODS: C-14 iodoantipyrine was used as a tracer to measure intrarenal blood flow in anaesthetised rats at multiple time points following nephrectomy. Autoradiographs were produced from tissue sections. C-14 concentrations were measured from standards thus allowing blood flow values to be calculated. RESULTS: Base line values for cortical and medullary blood flow were 806 +/- 63 and 373 +/- 39 ml./100 gm./min. (mean +/- SEM) respectively. At 2 hours post nephrectomy blood flow to both the cortex and medulla increased significantly (1152 +/- 54 and 594 +/- 37; p < 0.05). Blood flow had returned to control levels by 24 hours and was maintained at 5 days post-nephrectomy. Multiple discrete regions of high blood flow within the cortex were observed. Microautoradiography defined the morphological location of these discrete regions of higher blood flow as periglomerular vasculature. Diclofenac administration did not inhibit the augmentation in cortical blood flow post-nephrectomy, while medullary blood flow fell below base line values at both 30 minutes and 2 hours following nephrectomy. Sympathetic denervation did not affect the changes in cortical blood flow seen following nephrectomy, but did ameliorate the changes in medullary blood flow. CONCLUSIONS: Significant, transient changes in regional renal blood flow occur in the residual kidney following unilateral nephrectomy. The interaction between vasoactive mediators and the autonomic nervous system which produces changes in cortical blood flow is complex. It is evident, however, that medullary blood flow is dependent on local prostaglandin production and is also influenced by sympathetic nervous supply.

Animals↗