Archive of Medical Research

The sex chromosome that refused to die.

Chromosomes that harbor dominant sex determination loci are predicted to erode over time-losing genes, accumulating transposable elements, degenerating into a functional wasteland and ultimately becoming extinct. The Drosophila melanogaster Y chromosome is fairly far along this path to oblivion. The few genes on largely heterochromatic Y chromosome are required for spermatocyte-specific functions, but have no role in other tissues. Surprisingly, a recent paper shows that divergent Y chromosomes can substantially influence gene expression throughout the D. melanogaster genome.1 These results show that variation on Y has an important influence on the deployment of the genome. BioEssays 30:409-411, 2008. (c) 2008 Wiley Periodicals, Inc.

Malone JH, Oliver B.

Laboratory of Cellular and Developmental Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD.

Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia.

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD4+ Th1/Th2 and CD8+ T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity. (c) 2008 Wiley-Liss, Inc.

van den Hende M, van Poelgeest MI, van der Hulst JM, de Jong J, Drijfhout JW, Fleuren GJ, Valentijn AR, Wafelman AR, Slappendel GM, Melief CJ, Offringa R, van der Burg SH, Kenter GG.

Department of Gynecology, Leiden University Medical Center, Leiden, The Netherlands.

Estrogen-mediated downregulation of CD24 in breast cancer cells.

We have previously reported on the relevance of the prevalence of CD44(+)/CD24(-/low) cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative real-time PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ERalpha negative MDA-MB-231 cells suggesting that ERalpha was necessary. This was further confirmed by ERalpha silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ERalpha into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ERalpha corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ERalpha as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ERalpha and histone deacetylases. (c) 2008 Wiley-Liss, Inc.

Kaipparettu BA, Malik S, Konduri SD, Liu W, Rokavec M, van der Kuip H, Hoppe R, Hammerich-Hille S, Fritz P, Schroth W, Abele I, Das GM, Oesterreich S, Brauch H.

Division of Molecular Mechanisms of Origin and Treatment of Breast Cancer, Dr. Margarete Fischer‐Bosch‐Institute of Clinical Pharmacology, Stuttgart, Germany.

DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell lines.

Secreted frizzled-related protein 2 (sFRP2) is a negative modulator of the Wingless-type (Wnt) signaling pathway, and shown to be inactivated in renal cell carcinoma (RCC). However, the molecular mechanism of silencing of sFRP2 is not fully understood. Our study was designed to elucidate the silencing mechanism of sFRP2 in RCC. Expression of sFRP2 was examined in 20 pairs of primary cancers by immunohistochemistry. Kidney cell lines (HK-2, Caki-1, Caki-2, A-498 and ACHN) were analyzed for sFRP2 expression using real-time RT-PCR and Western blotting. The methylation status at 46 CpG sites of the 2 CpG islands in the sFRP2 promoter was characterized by bisulfite DNA sequencing. Histone modifications were assessed by chromatin immunoprecipitation (ChIP) assay using antibodies against AcH3, AcH4, H3K4 and H3K9. sFRP2 was frequently repressed in primary cancers and in RCC cells. The majority of sFRP2 negative cells had a methylated promoter. Meanwhile, sFRP2 expression was repressed by a hypomethylated promoter in Caki-1 cells, and these cells had a repressive histone modification at the promoter. In Caki-1 cells, sFRP2 was reactivated by trichostatin A (TSA). Repressive histone modifications were also observed in RCC cells with hypermethylated promoters, but sFRP2 was reactivated only by 5-aza-2\’-deoxycytidine (DAC) and not by TSA. However, the activation of the silenced sFRP2 gene could be achieved in all cells using a combination of DAC and TSA. This is the first report indicating that aberrant DNA methylation and histone modifications work together to silence the sFRP2 gene in RCC cells. (c) 2008 Wiley-Liss, Inc.

Kawamoto K, Hirata H, Kikuno N, Tanaka Y, Nakagawa M, Dahiya R.

Department of Urology, Veterans Affairs Medical Center and University of California School of Medicine, San Francisco, CA.

X-ray Burns-Painful, Protracted, and Preventable.

Very high doses of x-ray may produce deep burns in the backs of patients having fluoroscopically guided cardiac interventional procedures. While these incidents are uncommon they can be prevented by judicious limitation of fluoroscopy and timely repositioning of the x-ray tube. Better education and improved methods for dose mapping should make these distressing complications a thing of the past. Copyright (c) 2008 Wiley Periodicals, Inc.

Vlietstra RE, Wagner LK.

Cardiologist, Watson Clinic, Lakeland, Florida.

The Evolution of Management of Acute Coronary Syndromes (Unstable Angina and Non-ST Segment Elevation Myocardial Infarction): Part l.

As I advanced through medical school at Johns Hopkins, and throughout my subsequent training, I was constantly reminded of the contributions made to medicine by our predecessors. Many of whom were at Johns Hopkins, but many were not. As I continue active involvement in the education of medical students and physicians, it seems to me we have not done a good job of relating the history of how the modern management of patients with cardiovascular disease developed to our students, house staff, and fellows. Copyright (c) 2008 Wiley Periodicals, Inc.

Richard Conti C.

Fiber-bound nitrogen in gorilla diets: implications for estimating dietary protein intake of primates.

Protein is essential for living organisms, but digestibility of crude protein is poorly understood and difficult to predict. Nitrogen is used to estimate protein content because nitrogen is a component of the amino acids that comprise protein, but a substantial portion of the nitrogen in plants may be bound to fiber in an indigestible form. To estimate the amount of crude protein that is unavailable in the diets of mountain gorillas (Gorilla beringei) in Bwindi Impenetrable National Park, Uganda, foods routinely eaten were analyzed to determine the amount of nitrogen bound to the acid-detergent fiber residue. The amount of fiber-bound nitrogen varied among plant parts: herbaceous leaves 14.5+/-8.9% (reported as a percentage of crude protein on a dry matter (DM) basis), tree leaves (16.1+/-6.7% DM), pith/herbaceous peel (26.2+/-8.9% DM), fruit (34.7+/-17.8% DM), bark (43.8+/-15.6% DM), and decaying wood (85.2+/-14.6% DM). When crude protein and available protein intake of adult gorillas was estimated over a year, 15.1% of the dietary crude protein was indigestible. These results indicate that the proportion of fiber-bound protein in primate diets should be considered when estimating protein intake, food selection, and food/habitat quality. Am. J. Primatol. 70:1-5, 2008. (c) 2008 Wiley-Liss, Inc.

Rothman JM, Chapman CA, Pell AN.

Department of Animal Science, Cornell University, Ithaca, New York.

Factors influencing collagen biosynthesis.

The importance of collagen, the major structural protein of animal kingdom, in maintaining the normal structure and function of the skin is well known. The same property is exploited widely in medical and industrial fields in finding agents, which could influence the synthesis of this protein. In this context in vitro production of collagen is of high significance. A literature survey has been made to analyze the various factors that influence collagen biosynthesis. There are various physical and biological factors that can either induce or inhibit collagen biosynthesis at various levels of gene expression. However reports concentrating on the effects of plants-derived compounds in stimulating collagen synthesis are scanty. Since extracts of many plants are known to be beneficial in the wound healing process, plants-derived compounds will have a definite role in the regulation of collagen synthesis. The present study emphasizes the need for unearthing the role of these plant derived factors on collagen synthesis which will be of immense application in the medical field. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

Kavitha O, Thampan RV.

SAFI Institute of Advanced Study, Vazhayoor East PO, Malappuram District 673 633, Kerala, India.

Histone deacetylase 5 represses the transcription of cyclin D3.

Histone deacetylases (HDACs) modulate the transcription of a subset of genes by various means. HDAC5 is a class II HDAC whose subcellular location is signal-dependent. At present, its known gene targets are few in number. Here we identify a new HDAC5 target: the gene encoding the cell cycle-regulatory protein cyclin D3. When overexpressed in Balb/c-3T3 cells or mouse embryo fibroblasts, HDAC5 substantially reduced the activity of the cyclin D3 promoter and the abundance of endogenous cyclin D3 protein. Conversely, conditions that blocked HDAC5 function increased cyclin D3 expression: treatment of cells with the class I/II HDAC inhibitor trichostatin A (TSA), depletion of HDAC5 from cells by RNA interference, and cytoplasmic sequestration of HDAC5 by co-expression of catalytically active calcium/calmodulin-dependent protein kinase. HDAC5 interacted with the cyclin D3 promoter in vivo, and the HDAC5-responsive element was within 118 base pairs upstream of the transcription start site. Mutation of the Sp1 site and the cyclic AMP response element within this region did not affect the responsiveness of the cyclin D3 promoter to HDAC5 or TSA. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

Roy S, Shor AC, Bagui TK, Seto E, Pledger WJ.

Molecular Oncology Program, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, Florida 33612.

Epigenetics regulate centromere formation and kinetochore function.

The eukaryote centromere was initially defined cytologically as the primary constriction on vertebrate chromosomes and functionally as a chromosomal feature with a relatively low recombination frequency. Structurally, the centromere is the foundation for sister chromatid cohesion and kinetochore formation. Together these provide the basis for interaction between chromosomes and the mitotic spindle, allowing the efficient segregation of sister chromatids during cell division. Although centromeric (CEN) DNA is highly variable between species, in all cases the functional centromere forms in a chromatin domain defined by the substitution of histone H3 with the centromere specific H3 variant centromere protein A (CENP-A), also known as CENH3. Kinetochore formation and function are dependent on a variety of regional epigenetic modifications that appear to result in a loop chromatin conformation providing exterior CENH3 domains for kinetochore construction, and interior heterochromatin domains essential for sister chromatid cohesion. In addition pericentric heterochromatin provides a structural element required for spindle assembly checkpoint function. Advances in our understanding of CENH3 biology have resulted in a model where kinetochore location is specified by the epigenetic mark left after dilution of CENH3 to daughter DNA strands during S phase. This results in a self-renewing and self-reinforcing epigenetic state favorable to reliably mark centromere location, as well as to provide the optimal chromatin configuration for kinetochore formation and function. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

Gieni RS, Chan GK, Hendzel MJ.

Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2.