Oxidative stress and inflammation in lean and obese subjects with polycystic ovary syndrome

To determine whether polycystic ovary syndrome (PCOS) independently influences oxidative stress and inflammation or if the culprit is the comorbidities of obesity and/or insulin resistance common to this condition.

Polycystic ovary syndrome influences the level of serum amyloid A and activity of phospholipid transfer protein in HDL2 and HDL3

Insulin and insulin-like growth factor signaling increases proliferation and hyperplasia of the ovarian surface epithelium and decreases follicular integrity through upregulation of the PI3-kinase pathway

BACKGROUND: The ovarian surface epithelium responds to cytokines and hormonal cues to initiate proliferation and migration following ovulation. Although insulin and IGF are potent proliferative factors for the ovarian surface epithelium and IGF is required for follicle development, increased insulin and IGF activity are correlated with at least two gynecologic conditions: polycystic ovary syndrome and epithelial ovarian cancer. Although insulin and IGF are often components of in vitro culture media, little is known about the effects that these growth factors may have on the ovarian surface epithelium morphology or how signaling in the ovarian surface may affect follicular health and development.

METHODS: Ovaries from CD1 mice were cultured in alginate hydrogels in the presence or absence of 5 μg/ml insulin or IGF-I, as well as small molecule inhibitors of IR/IGF1R, PI 3-kinase signaling, or MAPK signaling. Tissues were analyzed by immunohistochemistry for expression of cytokeratin 8 to mark the ovarian surface epithelium, Müllerian inhibiting substance to mark secondary follicles, and BrdU incorporation to assess proliferation. Changes in gene expression in the ovarian surface epithelium in response to insulin or IGF-I were analyzed by transcription array. Extracellular matrix organization was evaluated by expression and localization of collagen IV.

RESULTS: Culture of ovarian organoids with insulin or IGF-I resulted in formation of hyperplastic OSE approximately 4-6 cell layers thick with a high rate of proliferation, as well as decreased MIS expression in secondary follicles. Inhibition of the MAPK pathway restored MIS expression reduced by insulin but only partially restored normal OSE growth and morphology. Inhibition of the PI 3-kinase pathway restored MIS expression reduced by IGF-I and restored OSE growth to a single cell layer. Insulin and IGF-I altered organization of collagen IV, which was restored by inhibition of PI 3-kinase signaling.

CONCLUSIONS: While insulin and IGF are often required for propagation of primary cells, these cytokines may act as potent mitogens to disrupt cell growth, resulting in formation of hyperplastic OSE and decreased follicular integrity as measured by MIS expression and collagen deposition. This may be due partly to altered collagen IV deposition and organization in the ovary in response to insulin and IGF signaling mediated by PI 3-kinase.

Further evidence consistent with Yqh as an indicator of risk of gonadal blastoma in Y-bearing mosaic Turner syndrome

An 8-year-old girl with some features of Turner syndrome and karyotype 45X/46XY had developed a bilateral gonadoblastoma in her rudimentary ovaries. Her normal Y chromosome showed the characteristic distal fluorescence, as seen in her father's. Another mosaic, this time 45X/46XidicY, and also with some Turner features had rudimentary ovaries, but no gonadoblastoma had developed at age 14. The nature of her idicY, which showed no fluorescent distal Yq and had one of the centromeres inactivated, was confirmed by in situ hybridisation with a Yp-specific probe. Using primers from a human Yp-specific sequence, we amplified DNA extracted from paraffin-embedded ovarian tissue from both cases, and from a normal testicle and a normal ovary as controls. The finding of the expected Y-derived PCR product in the rudimentary gonads from these mosaic patients indicates the presence of their Y chromosome in both. We discuss the validity of the findings, and the possible role of sequences in or near the fluorescent part of Yq in the origin of gonadoblastoma in Y-bearing mosaic Turner syndrome.

The role of cathepsin C in Papillon-Lefèvre syndrome, prepubertal periodontitis, and aggressive periodontitis

We have previously reported that loss-of-function mutations in the cathepsin C gene (CTSC) result in Papillon Lefevre syndrome, an autosomal recessive condition characterized by palmoplantar keratosis and early,onset, severe periodontitis. Others have also reported CTSC mutations in patients with severe prepubertal periodontitis, but without any skin manifestations. The possible role of CTSC variants in more common types of non-mendelian, early-onset, severe periodontitis ("aggressive periodontitis") has not been investigated. In this study, we have investigated the role of CTSC in all three conditions. We demonstrate that PLS is genetically homogeneous and the mutation spectrum that includes three novel mutations (c.386T>A/p. V129E, c.935A>G/p.Q312R, and c.1235A>G/p.Y412C) in 21 PLS families (including eight from our previous study) provides an insight into structure-function relationships of CTSC. Our data also suggest that a complete loss-of-function appears to be necessary for the manifestation of the phenotype, making it unlikely that weak CTSC mutations are a cause of aggressive periodontitis. This was confirmed by analyses of the CTSC activity in 30 subjects with aggressive periodontitis and age-sex matched controls, which demonstrated that there was no significant difference between these two groups (1,728.7 +/- SD 576.8 mu moles/mg/min vs. 1,678.7 +/- SD 527.2 mu moles/mg/min, respectively, p = 0.73). CTSC mutations were detected in only one of two families with prepubertal periodontitis; these did not form a separate functional class with respect to those observed in classical PLS. The affected individuals in the other prepubertal periodontitis family not only lacked CTSC mutations, but in addition did not share the haplotypes at the CTSC locus. These data suggest that prepubertal periodontitis is a genetically heterogeneous disease that, in some families, just represents a partially penetrant PLS. (C) 2004 Wiley-Liss, Inc.

Neurobiological model of visuo-spatial cognition in young Williams Syndrome children: measures of dorsal-stream and frontal function

We examine hypotheses for the neural basis of the profile of visual cognition in young children with Williams syndrome (WS). These are: (a) that it is a consequence of anomalies in sensory visual processing; (b) that it is a deficit of the dorsal relative to the ventral cortical stream; (c) that it reflects deficit of frontal function, in particular of fronto-parietal interaction; (d) that it is related to impaired function in the right hemisphere relative to the left. The tests reported here are particularly relevant to (b) and (c). They form part of a more extensive programme of investigating visual, visuospatial, and cognitive function in large group of children with WS children, aged 8 months to 15 years. To compare performance across tests, avoiding floor and ceiling effects, we have measured performance in children with WS in terms of the ‘age equivalence’ for typically developing children. In this paper the relation between dorsal and ventral function was tested by motion and form coherence thresholds respectively. We confirm the presence of a subgroup of children with WS who perform particularly poorly on the motion (dorsal) task. However, such performance is also characteristic of normally developingchildren up to 5 years: thus the WS performance may reflect an overall persisting immaturity of visuospatial processing which is particularly evident in the dorsal stream. Looking at the performance on the global coherence tasks of the entire WS group, we find that there is also a subgroup who have both high form and motion coherence thresholds, relative to the performance of children of the same chronological age and verbal age on the BPVS, suggesting a more general global processing deficit. Frontal function was tested by a counterpointing task, ability to retrieve a ball from a ‘detour box’, and the Stroop-like ‘day-night’ task, all of which require inhibition of a familiar response. When considered in relation to overall development as indexed by vocabulary, the day-night task shows little specific impairment, the detour box shows a significant delay relative to controls, and the counterpointing task shows a marked and persistent deficit in many children. We conclude that frontal control processes show most impairment in WS when they are associated with spatially directed responses, reflecting a deficit of fronto-parietal processing. However, children with WS may successfully reduce the effect of this impairment by verbally mediated strategies. On all these tasks we find a range of difficulties across individual children and a small subset of WS who show very good performance, equivalent to chronological age norms of typically developing children. Neurobiological models of visuo-spatial cognition in children with WS p.4 Overall, we conclude that children with WS have specific processing difficulties with tasks involving frontoparietal circuits within the spatial domain. However, some children with WS can achieve similar performance to typically developing children on some tasks involving the dorsal stream, although the strategies and processing may be different in the two groups.

Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.