Madeline Murphy

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.

BACKGROUND: Debate persists regarding the role of Desulfovibrio subspecies in ulcerative colitis. Combined microscopic and molecular techniques enable this issue to be investigated by allowing precise enumeration of specific bacterial species within the colonic mucous gel. The aim of this study was to combine laser capture microdissection and quantitative polymerase chain reaction to determine Desulfovibrio copy number in crypt-associated mucous gel in health and in acute and chronic ulcerative colitis. METHODS: Colonic mucosal biopsies were harvested from healthy controls (n = 19) and patients with acute (n = 10) or chronic (n = 10) ulcerative colitis. Crypt-associated mucous gel was obtained by laser capture microdissection throughout the colon. Pan-bacterial 16S rRNA and Desulfovibrio copy number/mm were obtained by polymerase chain reaction at each locus. Bacterial copy numbers were interrogated for correlation with location and disease activity. Data were evaluated using a combination of ordinary linear methods and linear mixed-effects models to cater for multiple interactions. RESULTS: Desulfovibrio positivity was significantly increased in acute and chronic ulcerative colitis at multiple levels within the colon, and after normalization with total bacterial signal, the relative Desulfovibrio load was increased in acute colitis compared with controls. Desulfovibrio counts did not significantly correlate with age, disease duration, or disease activity but interlevel correlations were found in adjacent colonic segments in the healthy control and chronic ulcerative colitis groups. CONCLUSION: The presence of Desulfovibrio subspecies is increased in ulcerative colitis and the data presented suggest that these bacteria represent an increased percentage of the colonic microbiome in acute ulcerative colitis.

The ovaries of 18 post-partum beef suckler cows were examined daily, using ultrasound, from Day 5 post partum until a normal oestrous cycle was completed. Periods of growth and regression of medium-sized (5-9 mm) follicles were identified before one medium follicle became dominant (single large follicle greater than or equal to 10 mm). The mean (+/- s.e.m.) number of days from parturition to detection of the first post-partum dominant follicle was 10.2 +/- 0.5. The first post-partum dominant follicle ovulated in 2/18 (11%) cows. The interval from calving to first ovulation (mean +/- s.e.m. = 35.9 +/- 3.3 days) was characterized by the growth and regression of a variable number (mean = 3.2 +/- 0.2; range 1-6) of dominant follicles. The maximum diameter of the dominant follicle increased as the cows approached first ovulation (P less than 0.05). Behavioural oestrus was not detected in 16/18 (89%) cows at first ovulation. Following first ovulation, the length of the subsequent cycle was short (mean = 9.7 +/- 0.5 days; range 8-15 days) in 14/18 (78%) cows and was characterized by the development and ovulation of a single dominant follicle. During oestrous cycles of normal length (mean = 20.6 +/- 0.5 days; range 18-23 days) one (N = 2), two (N = 7) or three (N = 8) dominant follicles were identified. The growth rate, maximum diameter or persistence of non-ovulatory dominant follicles before first ovulation or during oestrous cycles were not different (P greater than 0.05). These data show that, in beef suckler cows, follicular development and formation of a dominant follicle occur early after parturition and the incidence of ovulation of the first dominant follicle is low. The number of dominant follicles that develop before first ovulation is variable; first ovulation is rarely associated with oestrus and short cycles are common after first ovulation. It is concluded that prolonged anoestrus in post-partum beef suckler cows is due to lack of ovulation of a dominant follicle rather than delayed development of dominant follicles.

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High-grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR-433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR-433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p-Rb), and an increase in β-galactosidase activity. Furthermore, in silico analysis identified four possible miR-433 target genes associated with cellular senescence: cyclin-dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene. Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR-433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

Friesian x Hereford heifers (n = 19; mean +/- s.e.m. body weight (BW) = 375 +/- 5 kg) were used in a randomized incomplete block design. Heifers were fed 0.7 (n = 7; L), 1.1 (n = 7; M) or 1.8% (n = 5; G) of BW in dry matter (DM)/day for 10 weeks. Ovaries were examined by ultrasound, for one oestrous cycle, from week 5 of treatment. Maximum diameter of dominant follicles was smaller (P less than 0.05) in L (11.8 +/- 0.1 mm) than in M (13.7 +/- 0.2 mm) or G (13.2 +/- 0.3 mm) heifers. Growth rate (mm/day) of dominant follicles during the oestrous cycle was not affected (P greater than 0.05) by dietary intake. Persistence of dominant follicles was shorter (P less than 0.05) in L (9.8 +/- 0.2 days) than in M (11.9 +/- 0.3 days) or G (12.7 +/- 0.4 days) heifers. Three dominant follicles were identified during the oestrous cycle of 5 of 7 L, 3 of 7 M and 1 of 5 G heifers (P less than 0.10); 2 dominant follicles were identified in the remaining heifers (n = 2 of 7, 4 of 7 and 4 of 5, respectively). Length of the luteal phase and luteal-phase concentrations of progesterone were not affected (P greater than 0.05) by treatment. Low dietary intake reduced the diameter and persistence of dominant follicles during the oestrous cycle of beef heifers and tended to increase the proportion of oestrous cycles with 3 dominant follicles.