Anders Rehfeld

Alternative Polyadenylation of Tumor Suppressor Genes in Small Intestinal Neuroendocrine Tumors

The tumorigenesis of small intestinal neuroendocrine tumors (SI-NETs) is poorly understood. Recent studies have associated alternative polyadenylation (APA) with proliferation, cell transformation, and cancer. Polyadenylation is the process in which the pre-messenger RNA is cleaved at a polyA site and a polyA tail is added. Genes with two or more polyA sites can undergo APA. This produces two or more distinct mRNA isoforms with different 3′ untranslated regions. Additionally, APA can also produce mRNAs containing different 3′-terminal coding regions. Therefore, APA alters both the repertoire and the expression level of proteins. Here, we used high-throughput sequencing data to map polyA sites and characterize polyadenylation genome-wide in three SI-NETs and a reference sample. In the tumors, 16 genes showed significant changes of APA pattern, which lead to either the 3′ truncation of mRNA coding regions or 3′ untranslated regions. Among these, 11 genes had been previously associated with cancer, with 4 genes being known tumor suppressors: DCC, PDZD2, MAGI1, and DACT2. We validated the APA in three out of three cases with quantitative real-time-PCR. Our findings suggest that changes of APA pattern in these 16 genes could be involved in the tumorigenesis of SI-NETs. Furthermore, they also point to APA as a new target for both diagnostic and treatment of SI-NETs. The identified genes with APA specific to the SI-NETs could be further tested as diagnostic markers and drug targets for disease prevention and treatment.

Selection of high quality spermatozoa may be promoted by activated vitamin D in the woman.

ContextnThe vitamin D receptor (VDR) and enzymes involved in activation (CYP2R1, CYP27B1) and inactivation (CYP24A1) of vitamin D are expressed in ovary, testes, and spermatozoa.nnnObjectivenDetermine responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in spermatozoa from normal and infertile men, and identify the site of exposure and how 1,25(OH)2D3 influences sperm function.nnnDesignnSpermatozoa expressing VDR, CYP2R1, CYP27B1, and CYP24A1 were analyzed in normal and infertile men. 25-Hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D3], and 1,25(OH)2D3 were measured in serum, seminal fluid, cervical secretions, and ovarian follicular fluid. 1,25(OH)2D3 was tested on human spermatozoa.nnnSettingnTertiary center for fertility.nnnParticipantsnProtein expression in spermatozoa and semen quality were assessed in 230 infertile and 114 healthy men. Vitamin D metabolites were measured in fluids from 245 men and 13 women, while 74 oocytes and 17 semen donors were used for sperm-function tests.nnnMain Outcome MeasuresnVDR and CYP24A1 expressions in spermatozoa, fluid concentrations of 25-OHD, 24,25(OH)2D3, and 1,25(OH)2D3, and 1,25(OH)2D3-induced effects on intracellular calcium concentration ([Ca2+]i) and sperm-oocyte binding in vitro.nnnResultsnVDR and CYP24A1 were expressed in a >2-fold higher fraction of spermatozoa from normal than infertile men (P < 0.01). Concentrations of 25-OHD, 24,25(OH)2D, and 1,25(OH)2D3 were undetectable in seminal fluid but high in ovarian follicular fluid. Follicular concentrations of 1,25(OH)2D3 induced a modest increase in [Ca2+]i and sperm-oocyte binding in vitro (P < 0.05).nnnConclusionnPresence of VDR and CYP24A1 mainly in spermatozoa of higher quality supports that 1,25(OH)2D3 available in the female reproductive tract may promote selection of the best gametes for fertilization.

In vitro bioassay investigations of the endocrine disrupting potential of steviol glycosides and their metabolite steviol, components of the natural sweetener Stevia

The food industry is moving towards the use of natural sweeteners such as those produced by Stevia rebaudiana due to the number of health and safety concerns surrounding artificial sweeteners. Despite the fact that these sweeteners are natural; they cannot be assumed safe. Steviol glycosides have a steroidal structure and therefore may have the potential to act as an endocrine disruptor in the body. Reporter gene assays (RGAs), H295R steroidogenesis assay and Ca(2+) fluorimetry based assays using human sperm cells have been used to assess the endocrine disrupting potential of two steviol glycosides: stevioside and rebaudioside A, and their metabolite steviol. A decrease in transcriptional activity of the progestagen receptor was seen following treatment with 25,000 ng/ml steviol in the presence of progesterone (157 ng/ml) resulting in a 31% decrease in progestagen response (p=<0.01). At the level of steroidogenesis, the metabolite steviol (500-25,000 ng/ml) increased progesterone production significantly by 2.3 fold when exposed to 10,000 ng/ml (p=<0.05) and 5 fold when exposed to 25,000 ng/ml (p=<0.001). Additionally, steviol was found to induce an agonistic response on CatSper, a progesterone receptor of sperm, causing a rapid influx of Ca(2+). The response was fully inhibited using a specific CatSper inhibitor. These findings highlight the potential for steviol to act as a potential endocrine disruptor.

The Integumentary System

The skin is a large organ covering the exterior of the human body. It consists of two distinct layers: an epithelial barrier, the epidermis, and a strong and elastic layer of connective tissue, the dermis. The skin is attached to underlying structures by the subcutaneous tissue, which contains various amounts of adipose tissue. Associated with the skin are “epidermal derivatives”: hair follicles, nails, and sweat and sebaceous glands.

The Digestive System II: The Associated Organs

The salivary glands, the liver, the gallbladder, and the pancreas constitute the accessory organs of the digestive system. These organs have exocrine secretion that chemical breakdown ingested food to facilitate digestion. Furthermore, the liver and pancreas have multiple other functions essential for homeostasis, e.g., the endocrine function of the pancreas that regulates blood glucoses’ level and the detoxification function of the liver.

The Respiratory System

The respiratory system consists of conductive airways, paired lungs, and a ventilation mechanism. The respiration mechanism, an exchange of gases between inhaled air and the blood of the pulmonary capillaries, takes place in the alveoli. Apart from gas exchange, the respiratory system also filters and temperates inhaled air and contributes to production of speech (larynx), to sense of smell (nasal cavities), and to maintenance of the pH homeostasis (bicarbonate buffer system of the blood).

The Urinary System

The paired kidneys, the urine bladder, and the urethra constitute the urinary system. The kidneys remove waste products from the body, conserve water, and reabsorb electrolytes and metabolic substances. Furthermore, they are essential for maintenance of the pH homeostasis, the blood pressure, and the volume and composition of the extracellular fluid. The urine is transported from the kidneys to the urinary bladder via the paired ureters, and after storage in the urinary bladder, the urine is discharged via the urethra.

The Male Reproductive System

The male reproductive system consists of external reproductive organs, penis and scrotum, and internal reproductive organs: testes, a duct system, and accessory glands. The adult, paired testes produce both male sex hormones (androgens) and sperm cells (spermatogenesis). Androgens, primarily testosterone, are necessary for development and maintenance of male behavioral and physical manifestations and for spermatogenesis.

The Immune System and the Lymphatic Organs

The immune system is distributed throughout the entire human body and is composed of lymphatic cells, tissues, and organs as well as the lymphatic circulation and “nonspecific” defenses, e.g., the skin barrier. It is an essential defense against invading microorganisms, toxins, and foreign or altered cells. The primary effector cells of the immune system, lymphocytes are matured in the primary lymphatic organs and are activated in secondary lymphatic organs.

The Female Reproductive System

The female reproductive system consists of external reproductive organs, vulva, and internal sex organs: ovaries, uterine tubes, uterus, and vagina. During pregnancy a temporary organ, the placenta, plays an essential role.