Juan J. Bustamante

Molecular mechanisms underlying the health disparity of prostate cancer (PCa) have not been fully determined. In this study, we applied bioinformatic approach to identify and validate dysregulated genes associated with tumor aggressiveness in African American (AA) compared to Caucasian American (CA) men with PCa. We retrieved and analyzed microarray data from 619 PCa patients, 412 AA and 207 CA, and we validated these genes in tumor tissues and cell lines by Real-Time PCR, Western blot, immunocytochemistry (ICC) and immunohistochemistry (IHC) analyses. We identified 362 differentially expressed genes in AA men and involved in regulating signaling pathways associated with tumor aggressiveness. In PCa tissues and cells,NKX3.1, APPL2, TPD52, LTC4S, ALDH1A3andAMD1transcripts were significantly upregulated (p < 0.05) compared to normal cells. IHC confirmed the overexpression of TPD52 (p = 0.0098) and LTC4S (p < 0.0005) in AA compared to CA men. ICC and Western blot analyses additionally corroborated this observation in PCa cells. These findings suggest that dysregulation of transcripts in PCa may drive the disparity of PCa outcomes and provide new insights into development of new therapeutic agents against aggressive tumors. More studies are warranted to investigate the clinical significance of these dysregulated genes in promoting the oncogenic pathways in AA men.

Summary: Inositol hexakisphosphate (IP6) and inositol both regulate insulin secretion, but their combined use in the management of diabetes deserves investigation. The combined effects of IP6 and inositol supplementation were investigated in streptozotocin‐induced type 2 diabetic rats. The following groups of rats were studied for 8 weeks: non‐diabetic control, non‐diabetic high‐fat diet control, diabetic untreated, diabetic rats treated with the combination of IP6 and inositol (650 mg/kg bw) and diabetic rats treated with glibenclamide (10 mg/kg bw). High‐fat diet and streptozotocin were used to induce type 2 diabetes mellitus in Sprague–Dawley rats. Body weight, blood glucose, glycated haemoglobin, insulin, serum leptin, HOMA‐insulin resistance scores, intestinal amylase activity, serum and faecal lipids and food and fluid consumption were measured. Treatment with the combination significantly reduced blood glucose (306 ± 53 mg/dl) and insulin resistance score (1.93 ± 0.45) compared with diabetic controls (522 ± 24 mg/dl and 5.1 ± 0.69 respectively). Serum leptin (2.8 ± 0.6 ng/dl) and faecal triglycerides (108 ± 8 mg/dl) were significantly increased in rats treated with the combination compared with the diabetic control (1.8 ± 0.06 ng/dl and 86 ± 4 mg/dl). Serum triglyceride (47 ± 5.1 mg/dl), total cholesterol (98 ± 3.2 mg/dl) and food intake (26 ± 0.3 g) were significantly reduced by 45%, 25% and 25%, respectively, in rats treated with the combination compared with the diabetic control. Inositol and IP6 combined supplementation may be effective in the management of type 2 diabetes mellitus and related metabolic disorders by regulating some aspects of lipid and carbohydrate metabolism.

Drugs account for about 20% of gynecomastia cases in men. As a number of factors can alter the estrogen:androgen ratio, several pathophysiologic mechanisms are associated with drugs causing this disorder. Antiandrogens, protease inhibitors, and nucleoside reverse transcriptase inhibitors are the most common drug causes of gynecomastia, whereas first‐generation antipsychotics, spironolactone, verapamil, and cimetidine are less common causes. Other drugs have been reported rarely as causes. Treatment may involve switching to an alternative agent or may require surgery or irradiation if the causative agent cannot be discontinued. We reviewed the literature on drug‐induced gynecomastia and provided another perspective by reviewing data from the United States Food and Drug Administration's A dverse E vent R eporting S ystem. Epidemiologic studies are needed to provide a more accurate description of the frequency of drug‐induced gynecomastia.

Pregnancy is characterized by physiological adjustments in the maternal compartment. In this investigation, the influence of pregnancy on maternal liver was examined in CD-1 mice. Dramatic changes were observed in the size of the maternal liver during pregnancy. Livers doubled in weight from the non-pregnant state to day 18 of pregnancy. The pregnancy-induced hepatomegaly was a physiological event of liver growth confirmed by DNA content increase and detection of hepatocyte hyperplasia and hypertrophy. Growth of the liver was initiated following implantation and peaked at parturition. The expression and/or activities of key genes known to regulate liver regeneration, a phenomenon of liver growth compensatory to liver mass loss, were investigated. The results showed that pregnancy-dependent liver growth was associated with interleukin (IL)-6, tumor necrosis factor α, c-Jun and IL-1 β, but independent of hepatocyte growth factor, fibroblast growth factor 1, tumor necrosis factor receptor 1, constitutive androstane receptor and pregnane X receptor. Furthermore, maternal liver growth was associated with the activation of hepatic signal transducer and activator of transcription 3, β-catenin and epidermal growth factor receptor, but pregnancy did not activate hepatic c-Met. The findings suggest that the molecular mechanisms regulating pregnancy-induced liver growth and injury-induced liver regeneration exhibit overlapping features but are not identical. In summary, the liver of the mouse adapts to the demands of pregnancy via a dramatic growth response driven by hepatocyte proliferation and size increase.

Background: Maternal metabolic demands change dramatically during the course of gestation and must be co‐ordinated with the needs of the developing placenta and fetus. The liver is critically involved in metabolism and other important functions. However, maternal hepatic adjustments to pregnancy are poorly understood. Aim: The aim of the study was to evaluate the influences of pregnancy on the maternal liver growth and gene expression profile. Methods: Holtzman Sprague–Dawley rats were mated and sacrificed at various stages of gestation and post‐partum. The maternal livers were analysed in gravimetric response, DNA content by PicoGreen dsDNA quantitation reagent, hepatocyte ploidy by flow cytometry and hepatocyte proliferation by ki‐67 immunostaining. Gene expression profiling of non‐pregnant and gestation d18.5 maternal hepatic tissue was analysed using a DNA microarray approach and partially verified by northern blot or quantitative real‐time PCR analysis. Results: During pregnancy, the liver exhibited approximately an 80% increase in size, proportional to the increase in body weight of the pregnant animals. The pregnancy‐induced hepatomegaly was a physiological event of liver growth manifested by increases in maternal hepatic DNA content and hepatocyte proliferation. Pregnancy did not affect hepatocyte polyploidization. Pregnancy‐dependent changes in hepatic expression were noted for a number of genes, including those associated with cell proliferation, cytokine signalling, liver regeneration and metabolism. Conclusions: The metabolic demands of pregnancy cause marked adjustments in maternal liver physiology. Central to these adjustments are an expansion in hepatic capacity and changes in hepatic gene expression. Our findings provide insights into pregnancy‐dependent hepatic adaptations.

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc₂, N‐acetyl galactosamine₁, Gal₁). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.

Background/Aims: During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia‐inducible factor‐1α (HIF‐1α), is critical for upregulation of profibrotic mediators, such as platelet‐derived growth factor‐A (PDGF‐A), PDGF‐B and plasminogen activator inhibitor‐1 (PAI‐1) in the liver, during the development of fibrosis. What remains unknown is the cell type‐specific regulation of these genes by HIF‐1α in liver cell types. Accordingly, the hypothesis was tested that HIF‐1α is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells.Methods: In this study, hepatocytes were isolated from the livers of control and HIF‐1α‐ or HIF‐1β‐deficient mice and exposed to hypoxia.Results: Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF‐1α and upregulated PAI‐1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin‐1 (ADM‐1) and ADM‐2. In contrast, the levels of PDGF‐A and PDGF‐B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF‐1α‐deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia‐regulated transcription factors, such as HIF‐2α, may also regulate these genes. In support of this, HIF‐2α was activated in hypoxic hepatocytes, and exposure of HIF‐1β‐deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI‐1, vascular endothelial cell growth factor and ADM‐1, suggesting that HIF‐2α may also contribute to upregulation of these genes in hypoxic hepatocytes.Conclusions: Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.

The membrane‐bound rat GH‐R and an alternatively spliced isoform, the soluble rat GH‐BP, are comprised of identical N‐terminal GH‐binding domains; however, their C‐terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C‐terminal peptide sequence of the rat GH‐BP (GH‐BP_263–279) was synthesized and used as an immunogen in rabbits. Solid‐phase peptide synthesis of four GH‐BP_263–279 segments onto a tetravalent Lys₂‐Lys‐β‐Ala‐OH core peptide was carried out using Fmoc chemistry. The mass of the RP‐HPLC‐purified synthetic product, 8398 Da, determined by ESI‐MS, was identical to expected mass. Three anti‐rat GH‐BP_263–279 MAP antisera, BETO‐8039, BETO‐8040, and BETO‐8041, at dilutions of 10⁻³, recognized both the rat GH‐BP_263–279 MAP and recombinant mouse GH‐BP with ED₅₀s within a range of 5–10 fmol, but did not cross‐react with BSA in dot blot analyses. BETO‐8041 antisera (10⁻³ dilution) recognized GH‐BPs of rat serum and liver having M_rs ranging from 35 to 130 kDa, but did not recognize full‐length rat GH‐Rs. The antisera also detected recombinant mouse GH‐BPs. In summary, the tetravalent rat GH‐BP_263–279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C‐termini of both rat and mouse GH‐BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH‐BPs. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.