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Pathogen research abs 1 || Pathogen research abs 2 || Pathogen research abs 3 || Pathogen research abs 4 || Pathogen research abs 5 || Hormone and endocrine research abs 1 || Hormone and endocrine research abs 2 || Hormone and endocrine research abs 3 || Hormone and endocrine research abs 4 || Hormone and endocrine research abs 5 || Follicle and follicular cells research abs 1 || Interferon research abs 1 || Hemoglobin research abs || Stem cell research abs || Nucleic acid research abs

Endocrinology. 2000 Oct;141(10):3737-48.
Promoter elements and transcription factors involved in differentiation-dependent human chorionic gonadotrophin-alpha messenger ribonucleic acid expression of term villous trophoblasts.

Knofler M, Saleh L, Bauer S, Vasicek R, Griesinger G, Strohmer H, Helmer H, Husslein P.

Department of Obstetrics and Gynecology, University of Vienna, Austria. martin.knoeflekh-wien.ac.at

Differentiation of primary villous cytotrophoblasts into syncytia is associated with increasing production of alpha and beta human CG subunits, which is predominantly governed at the level of messenger RNA expression. Here, we present a detailed study on the mechanisms involved in the differentiation-dependent regulation of the trophoblast-specific CGalpha gene promoter. Site-directed mutations in each of the five DNA-elements of the composite enhancer were performed to investigate the contribution of the individual regulatory sequences to the overall transcriptional activity of the promoter at two different stages of trophoblast in vitro differentiation. We show that deletion of one cyclic AMP response element (CRE) did not affect CGalpha promoter activity in cytotrophoblasts; however, it reduced transcription by 33% in differentiating cultures. Removal of both CREs almost abolished transcription at early and later stages of in vitro differentiation. Upon mutation the enhancer elements alphaACT, JRE, and CCAAT significantly decreased luciferase reporter transcription; however their contribution to the total promoter activity did not change during in vitro differentiation. Contrary to that, mutated TSE diminished promoter activity by 19% during 12 and 48 h of cultivation but reduced luciferase expression by 78% between 48 and 84 h of differentiation. In electrophoretic mobility shift assay, the TSE interacted with activating protein (AP)-2alpha in both primary trophoblasts and choriocarcinoma cells. While CRE-interacting proteins were detectable 12 h after isolation, the TSE-binding complex did not appear before 36 h of in vitro differentiation. During syncytium formation increasing protein expression of activating transcription factor (ATF)-1, cAMP response element-binding protein (CREB)-1, and AP-2alpha was observed on Western blots. Moreover, phosphorylated CREB-1 and ATF-1 accumulated between 24 and 78 h of trophoblast cultivation. By fluorescence immunohistochemistry, we show that CREB-1 was predominantly expressed in syncytiotrophoblasts, whereas ATF-1 and AP-2alpha localized to the syncytium and some cytototrophoblasts as well as to stromal and endothelial cells of the placental villus. Phosphorylated CREB-1/ATF-1 and the coactivator protein CBP were primarily detected in syncytial nuclei, suggesting the presence of functional, cAMP-dependent transcriptional complexes in the differentiated tissue. In agreement to the in vivo situation, phosphorylated CREB-1/ATF-1 were observed in nuclei of the differentiated trophoblast cultures. The activity of the CGalpha promoter as well as CREB-1/ATF-1 phosphorylation increased upon elevation of cAMP levels and overexpression of the catalytic subunit of protein kinase A. Additionally, we demonstrate that overproduction of the enzyme enhanced protein expression and binding of AP-2alpha to the TSE. We conclude that differentiation-dependent transcription of the CGalpha gene in villous trophoblasts is mainly governed by increasing expression of AP-2alpha and PKA-dependent phosphorylation of CREB-1 and ATF-1.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11014230&dopt=Abstract

Endocrinology. 2000 Oct;141(10):3821-31.
A novel messenger ribonucleic acid homologous to human MAGE-D is strongly expressed in rat Sertoli cells and weakly in Leydig cells and is regulated by follitropin, lutropin, and prolactin.

Hennuy B, Reiter E, Cornet A, Bruyninx M, Daukandt M, Houssa P, N'Guyen VH, Closset J, Hennen G.

Biochemistry and Laboratory of Endocrinology, Institute of Pathology, University of Liege, Belgium.

We have cloned a novel complementary DNA whose expression was decreased in rat Sertoli cell cultures after treatment with FSH. This complementary DNA encodes a protein of 570 amino acids and shares 92% homology with the human MAGE-D protein. In contrast to other MAGE genes (A, B, or C), we have shown that MAGE-D expression was ubiquitous in healthy rat tissues. In the seminiferous tubules, the MAGE-D was expressed in Sertoli cells but not in germ cells as demonstrated by RT-PCR and in situ hybridization, whereas for the other MAGE genes, expression has been shown to be restricted to germ cells. Interestingly, MAGE-D was also detected for the first time in the female gonad by Northern blotting. In MLTC-1 cells (mouse Leydig tumor cell line-1), LH and PRL stimulated MAGE-D expression. Using hypophysectomized rats, it was confirmed that FSH decreased MAGE-D expression, whereas LH and PRL increased MAGE-D messenger RNA level in the whole testis most probably through a direct action on Leydig cells. As MAGE-D is present in both the seminiferous compartment and interstitium and hormonally regulated in each, it is possible that it has specific functions in each compartment during the development and the maintenance of the testis.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11014239&dopt=Abstract

Biochemistry. 2000 Oct 10;39(40):12113-20.
Metal-phosphate interactions in the hammerhead ribozyme observed by 31P NMR and phosphorothioate substitutions.

Maderia M, Hunsicker LM, DeRose VJ.

Department of Chemistry, Texas A&M University, College Station, Texas 77842, USA.

The hammerhead ribozyme is a catalytic RNA that requires divalent metal cations for activity under moderate ionic strength. Two important sites that are proposed to bind metal ions in the hammerhead ribozyme are the A9/G10.1 site, located at the junction between stem II and the conserved core, and the scissile phosphate (P1.1). (31)P NMR spectroscopy in conjunction with phosphorothioate substitutions is used in this study to investigate these putative metal sites. The (31)P NMR feature of a phosphorothioate appears in a unique spectral window and can be monitored for changes upon addition of metals. Addition of 1-2 equiv of Cd(2+) to the hammerhead with an A9-S(Rp) or A9-S(S)(Rp) substitution results in a 2-3 ppm upfield shift of the (31)P NMR resonance. In contrast, the P1.1-S(Rp) and P1.1-S(Sp) (31)P NMR features shift slightly and in opposite directions, with a total change in delta of </=0.6 ppm with addition of up to 10 equiv of Cd(2+). No significant shifts are observed for an RNA.RNA duplex with a single, internal phosphorothioate modification upon addition of Cd(2+). Data obtained using model compounds including diethyl phosphate/thiophosphate, AMP, and AMPS, show that a Cd(2+)-S interaction yields an upfield shift for the (31)P NMR resonance, even in the case of a weak coordination such as with diethyl thiophosphate. Taken together, these data predict that Cd(2+) has a high affinity for the A9 site and suggest that there is flexibility in metal coordination within the binding pocket. Cd(2+) interactions with the cleavage site P1.1-S positions are weaker and appear to be stereospecific. These data have implications for mechanisms that have been proposed to explain the influence of metal ions on hammerhead ribozyme activity. These experiments also show the potential utility of (31)P NMR spectroscopy in conjunction with phosphorothioates as a probe for metal binding sites in nucleic acids.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11015188&dopt=Abstract

J Clin Microbiol. 2000 Oct;38(10):3656-62.
Genotypic determination of Mycobacterium tuberculosis antibiotic resistance using a novel mutation detection method, the branch migration inhibition M. tuberculosis antibiotic resistance test.

Liu YP, Behr MA, Small PM, Kurn N.

Advanced Diagnostics Division, Dade Behring Inc., San Jose, California 95135, USA. yenping_liadebehring.com

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413-424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in M. tuberculosis, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426-5430, 1994). RMP and PZA resistances are associated with multiple mutations within the rpoB and pncA genes, respectively. M. tuberculosis genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a "blind study." Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of M. tuberculosis clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11015379&dopt=Abstract

J Clin Microbiol. 2000 Oct;38(10):3860-3.
Comparison of six commercial DNA extraction kits for recovery of cytomegalovirus DNA from spiked human specimens.

Fahle GA, Fischer SH.

Microbiology Service, Clinical Pathology Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892-1508, USA. gfahlih.gov

We evaluated six commercially available DNA extraction kits for their ability to recover DNA from various dilutions of cytomegalovirus (CMV) added to four different specimens: bronchoalveolar lavage, cerebral spinal fluid, plasma, and whole blood. The kits evaluated included the Puregene DNA isolation kit (PG), Generation Capture Column kit, MasterPure DNA purification kit, IsoQuick nucleic acid extraction kit, QIAamp blood kit, and NucliSens isolation kit (NS). All six kits evaluated effectively removed PCR inhibitors from each of the four specimen types and produced consistently positive results down to a spiked concentration of 200 PFU of whole CMV per ml. However, the NS and PG resulted in the most consistently positive results at the lowest concentrations of spiked CMV (4 and 0.4 PFU/ml) and, in this evaluation, offered the most sensitive methods for extracting CMV DNA from the four different spiked specimens. Processing time and cost were also evaluated.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11015421&dopt=Abstract

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