Multiplex fluorescence in situ hybridization identifies novel rearrangements of chromosomes 6, 15, and 18 in primary uveal melanoma.

Uveal melanomas are the commonest ocular tumour of adults and are characterized by reproducible alterations of chromosomes 1, 3, 6 and 8. These alterations are of prognostic relevance and have also be shown to correlate to high risk and low risk metastatic categories of uveal melanoma as defined by micro-array analysis. It is, however, possible that a catalogue of relevant genetic alterations, involving gene rearrangement rather than amplification, have as yet eluded identification.

Components of metabolic syndrome and coronary artery disease in female Ossabaw swine fed excess atherogenic diet.

Ossabaw swine have a 'thrifty genotype' (propensity to obesity) that enables them to survive seasonal food shortages in their native environment. Consumption of excess kcal causes animals of the thrifty genotype to manifest components of the metabolic syndrome, including central (intra-abdominal) obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. We determined whether female Ossabaw swine manifest multiple components of the metabolic syndrome by comparing lean pigs fed a normal maintenance diet (7% kcal from fat; lean, n = 9) or excess chow with 45% kcal from fat and 2% cholesterol (obese, n = 8).

B cell-deficient NOD.H-2h4 mice have CD4+CD25+ T regulatory cells that inhibit the development of spontaneous autoimmune thyroiditis.

Wild-type (WT) NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) when given 0.05% NaI in their drinking water, whereas B cell-deficient NOD.

Cyclooxygenase-2 regulation of the age-related decline in testosterone biosynthesis.

The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis.

HER2 amplification status in breast cancer: a comparison between immunohistochemical staining and fluorescence in situ hybridisation using manual and automated quantitative image analysis scoring techniques.

Aims: To compare the results of breast cancer sections with HercepTesttrade mark immunohistochemistry (IHC) scores ranging from 0 to 3+ with fluorescence in situ hybridisation (FISH) for HER2 amplification. The HER2 digital scoring application of the Micrometastasis Detection System (MDS) was used, together with manual scoring of FISH and HercepTest, to determine whether this system provides an accurate alternative.

Methods: Paraffin wax embedded sections were stained using HercepTest and analysed by eye and automated quantitative image analysis.

MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2.

ERK and p38 MAP kinases, acting through the downstream mitogen- and stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes--including those at Fos and Jun--concomitant with gene induction. S10 and S28 on the H3 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear with similar time-courses and both occur on H3 tails that are highly sensitive to TSA-induced hyperacetylation, similarities which might suggest that MSK1/2 phosphorylates both sites on the same H3 tails.

A retrospective study of growth hormone use in adolescents with cystic fibrosis.

Objectives: Studies of growth hormone (GH) effectiveness in prepubertal children with cystic fibrosis (CF) have been published previously. We present a retrospective study of GH treatment in adolescents with CF.

Study Design: We performed a retrospective evaluation of data from 25 pubertal adolescents (ages 13 years 4 months to 16 years 11 months, Tanner stage III or IV).

Heat shock, histone H3 phosphorylation and the cell cycle.

Genome-wide and gene-specific changes in histone H3 phosphorylation during heat shock have recently been described using two well-established experimental models, the "puffing" of heat shock loci in Drosophila polytene chromosomes and the induction of hsp70 mRNA transcripts in cultured mouse cells. Despite conservation of the molecular participants and overall stress response in these two organisms, some striking differences have emerged. Here, we summarize accounts of heat shock-modulated histone phosphorylation in Drosophila and mouse cells highlighting these differences.

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression.

Background: In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners.

Perceived stress and cortisol levels predict speed of wound healing in healthy male adults.

The main purpose of the present study was to investigate the association between perceived stress and impaired cutaneous wound healing in humans using a novel wound assessment technique, and taking into account putative mediating factors such as cortisol levels, health behaviours, and personality factors. The study made use of a prospective, within-subjects design in which 24 male non-smokers participated. Every subject received a standard 4mm-punch biopsy, and the healing progress was monitored via high-resolution ultrasound scanning.

Detection of cryptic MLL insertions using a commercial dual-color fluorescence in situ hybridization probe.

Involvement of the MLL gene located at chromosome region 11q23 is a frequent occurrence in both acute myelocytic leukemia and acute lymphoblastic leukemia. More than 30 loci have now been associated with MLL, usually by reciprocal translocation. Deletions, insertions, and more complex rearrangements of MLL are rarely seen.

Involvement of 5-lipoxygenase metabolites of arachidonic acid in cyclic AMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression.

To understand the mechanism for the role of arachidonic acid (AA) in steroidogenic acute regulatory (StAR) gene transcription, sections of the -1/-966 StAR promoter were deleted to produce constructs of -1/-426, -1/-211, -1/-151, and -1/-110 and inserted into the PGL3 vector to drive luciferase expression. Results indicated that -1/-151 StAR promoter contains the elements that are most responsive to AA. Electrophoretic mobility shift assays using nuclear extracts from AA-treated MA-10 Leydig tumor cells showed that AA enhanced specific binding of the nuclear extract to a 30bp (-67/-96) sequence of the StAR promoter.

Inhibition of cyclooxygenase-2 activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.

To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mM dibutyryl cAMP (Bt(2)cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 microM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.

MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of histone H3 and HMG-14.

Cells respond to mitogenic or stress stimuli by the rapid induction of immediate-early (IE) genes, which occurs concomitantly with the phosphorylation of histone H3 and the high-mobility-group protein HMG-14. In mammalian cells this response is mediated via ERK and p38 MAP kinase pathways, but the identity of the downstream kinase that phosphorylates histone H3 has been contentious. One study, based on Coffin- Lowry cells defective in RSK2, reported that RSK2 was the histone H3 kinase, while a second study, based on the efficiency of RSKs and MSKs as in vitro histone H3 kinases, and their relative susceptibility to kinase inhibitors, suggested that MSKs were responsible.

Ovarian follicle populations of the rat express TGF-beta signalling pathways.

The transforming growth factor-beta (TGF-beta) superfamily comprises more than 40 members, classified on the basis of structural similarity. These factors elicit a diverse range of cellular responses in insects, nematodes and vertebrates, via serine/threonine kinase receptors and intracellular Smad proteins, which when activated mediate gene transcription. Some members of the superfamily, notably activin, TGF-beta, GDF-9 and the bone morphogenetic proteins have been shown to influence ovarian function.

The effect of 880 nm low level laser energy on human fibroblast cell numbers: a possible role in hypertrophic wound healing.

Low level lasers (LLLs) have been shown to induce therapeutic effects in wound healing. However, there have been few LLL studies on burn wounds which may become unsightly, hypertrophic and impair function. Inhibitory effects on the healing of fibrotic wounds, prone to hypertrophy may be expected to reasonably reduce the problems accompanying hypertrophic scarring.

Hepatitis B virus core antigen: enhancement of its production in Escherichia coli, and interaction of the core particles with the viral surface antigen.

The core antigen (HBcAg) of hepatitis B Virus (HBV) can be expressed in Escherichia coil where it assembles into icosahedral particles containing 240 or 180 subunits. Analysis of the two kinds of particles by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a substantial proportion of their subunits were smaller than the full-length HBcAg monomer and of variable size, but all had the same N-terminal sequence showing that the smaller species were heterogeneous in their arginine-rich C-terminal regions. Around 50% of these arginine residues are encoded by the triplet AGA which is rare in E.

Wound healing assessment using 20 MHz ultrasound and photography.

Background/aims: To compare two non-invasive techniques of assessing wound healing, photography and high resolution ultrasound (HRUS) scanning, in experimentally induced full-thickness human skin wounds.

Methods: Punch biopsy wounds, 4 mm in diameter, were made aseptically through locally anaesthetised skin on the anterior (volar) surface of the non-dominant forearm, 3 cm below the base of the cubital fossa, of 20 human participants. The wounds were treated with a topical antibiotic and covered for 3 days with Mepore sterile dressings.

Recruitment and development of the follicle; the roles of the transforming growth factor-beta superfamily.

Peripheral endocrine hormones and local paracrine and autocrine factors contribute, in a coordinated fashion, to the processes of recruitment, development or atresia, selection and ovulation of follicles. Among the local ovarian factors, there is growing evidence from genetic and experimental data that many members of the transforming growth factor (TGFbeta) superfamily have a biological role to play in folliculogenesis. These members include activin, inhibin, TGFbeta, BMP, GDF9 and perhaps MIS.

Ovarian steroid receptors and their role in ovarian function.

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The precise role of oestrogen in the ovary remains to be elucidated, hence the data presented here which arises from studies designed to resolve this issue. Oestrogens signal via two receptor subtypes ERalpha and ERbeta, both of which are present in the ovary.

Interaction between arachidonic acid and cAMP signaling pathways enhances steroidogenesis and StAR gene expression in MA-10 Leydig tumor cells.

Previous studies have demonstrated that trophic hormone stimulation induced cyclic AMP (cAMP) formation and arachidonic acid (AA) release from phospholipids and that both these compounds were required for steroid biosynthesis and steroidogenic acute regulatory (StAR) gene expression in MA-10 mouse Leydig tumor cells. The present study further investigates the synergistic effects of the AA and cAMP interaction on steroidogenesis. To demonstrate cAMP-induced AA release, MA-10 cells were pre-loaded with 3H-AA and subsequently treated with dibutyryl cyclic AMP (dbcAMP).

Expression and localization of activin receptors, Smads, and beta glycan to the postnatal rat ovary.

Despite understanding the molecular basis of activin/TGF beta and bone morphogenetic protein (BMP) signaling, this study is the first to characterize multiple, sequential elements of these pathways in the ovary concurrently. The expression of activin/BMP receptor, Smad, and beta glycan mRNAs by postnatal rat ovaries were investigated by real-time PCR. Activin/BMP receptors (ActRIA, ActRIB, ActRIIA, and ActRIIB), beta glycan, and Smad 1-8 mRNAs were expressed by the ovary.

The ovarian phenotype of the aromatase knockout (ArKO) mouse.

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone.

Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family.

The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression.

Growth hormone improves clinical status in prepubertal children with cystic fibrosis: results of a randomized controlled trial.

Objectives: We conducted a 1-year randomized controlled trial to test the hypothesis that growth hormone (GH) improves the clinical status of children with cystic fibrosis.

Study Design: Nineteen prepubertal children were randomized to control (NonTX, n = 9) or to daily injections of GH (0.3 mg/kg/wk) (GHTX, n = 10) for 1 year.