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Biomedical subjects

J Stephan

Publications and source records attributed to J Stephan.

6 recordsLinked to original sources
PubMed2003

Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells.

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R-GFP overexpressing hVSMCs, Ang II (10(-9) and 10(-4) M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1R-GFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1R-GFP overexpressing hVSMCs, Ang II (10(-15) to 10(-4) M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 x 10(-11) and 5 x 10(-9) M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.

Aniline Compounds↗
PubMed2001

Highly efficient single molecule detection in microstructures.

For DNA single molecule sequencing, the complete detection of all dye-labeled monomers which are cleaved off during the sequencing reaction is an essential requirement. In this work we address the feasibility of single molecule detection in microstructures with a confocal multi element set-up. We present statistical data on single molecule recognition and explain a refined data evaluation technique for single molecule burst analysis. From these data the signal-to-noise ratio in microstructures is evaluated as well as the overall detection efficiency. So far, detection efficiencies of single molecule events of up to 60% have been shown in microstructures.

Biochemistry↗
PubMed2001

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Biochemistry↗
PubMed2001

Human papillomavirus infection at the United States-Mexico border: implications for cervical cancer prevention and control.

The United States-Mexico border is a region comprised of a country with one of the highest rates of invasive cervical cancer (Mexico) and a country with one of the lowest rates (United States). Recent evidence clearly indicates that human papillomavirus (HPV) infection is the cause of cervical cancer. The distribution of specific types of HPV is known to vary in different regions of the world, as do the cofactors that may inhibit or promote HPV carcinogenesis. Estimating the prevalence of oncogenic HPV is needed for guiding vaccine development. The purpose of this study was to determine the prevalence of oncogenic and nononcogenic HPV types and risk factors for HPV among women residing along the United States-Mexico border. A cross-sectional study of 2319 women, ages 15-79 years, self-referring for gynecological care was conducted between 1997 and 1998. HPV was detected by PCR using the PYGMY 09/11 L1 consensus primer, and HPV genotyping was conducted using the reverse line blot method. Overall, the HPV prevalence was 14.4% with no significant differences observed by country after adjustment for age. HPV 16 was the most commonly detected HPV type in both the United States and Mexico. Among women with high-grade squamous intraepithelial lesions, HPV types 58, 45, 51, 31, 35, 55, and 73 were most common in Mexico, and HPV types 18, 31, 35, 51, 52, and 58 were most common in the United States. In both countries, HPV prevalence declined linearly with age from 25% among women ages 15-19 years to 5.3% among women 56-65 years. Factors significantly independently associated with HPV infection were older age [adjusted odds ratio (AOR) = 0.15 for ages 56-65 years compared with those 15-19 years], a marital status other than married (AOR = 1.58-3.29), increased numbers of lifetime male partners (AOR = 3.8 for > or =10 partners compared with 1 partner), concurrent infection with Chlamydia trachomatis (AOR = 1.79), ever use of Norplant (AOR = 2.69), and current use of injectable contraceptives (AOR = 2.29). Risk factors for HPV infection did not differ by country. Results from this study suggest that in addition to HPV 16 and 18, HPV types 31, 45, 51, and 58 should be considered for inclusion in an HPV prevention vaccine for distribution in Mexico.

Adolescent↗