ToothPrint, a proteomic database for dental tissues.

Increasing demand exists to disseminate and integrate proteomic data as proteome analysis assumes a commanding role in the postgenome era. Databases on the World Wide Web are an effective means to share information obtained from two-dimensional gels and allied proteomic approaches. Here we report the establishment of ToothPrint, a proteomic database for dental tissues accessed at http://toothprint.otago.ac.nz. Using developing rat enamel as a prototype, ToothPrint provides a variety of functionally relevant data (ligand binding, subcellular localisation, developmental regulation) in addition to protein identification maps. Features designed to enhance usability of the website and simplify its computing requirements are also outlined. Customized for mineralizing tissues, ToothPrint should prove to be an effective bioinformatic resource for investigations of dental biology.

Evaluation of the NucliSens Basic Kit for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genital tract specimens using nucleic acid sequence-based amplification of 16S rRNA.

We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of C. trachomatis and N. gonorrhoeae and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of C. trachomatis, 1 CFU of N. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for N. gonorrhoeae by culture and NASBA and a total of 96 specimens for C. trachomatis by PCR and NASBA. The Basic Kit detected 139 of 142 N. gonorrhoeae culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For C. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both N. gonorrhoeae and C. trachomatis, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.

Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity.

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and -70 degreesC overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or -70 degreesC and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.

Transcriptional activation by DNA-binding derivatives of HSV-1 VP16 that lack the carboxyl-terminal acidic activation domain.

The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex with cellular components Oct-1 and VCAF-1, contributing a potent carboxyl-terminal acidic activation domain that is essential for activation of gene expression in mammalian cells. We show here that VP16, devoid of this acidic activation domain, functions as a strong transcriptional activator in the yeast Saccharomyces cerevisiae when appended onto a heterologous GAL4 DNA binding domain, as determined by measuring activation of a resident GAL1:lacZ reporter gene. Deletion analysis indicated that sequences contained within the amino-terminal 369 amino acids of VP16 were necessary for transactivation by truncated VP16. Activation by truncated VP16 in yeast was comparable to that observed with a hybrid protein consisting of the GAL4 DNA binding domain linked to the VP16 acidic activation domain. Similar GAL4-VP16 hybrid proteins were only marginally active in mammalian cells. Sequence requirements for transactivation by truncated VP16 can be demarcated from domains of VP16 that are required for interaction with VCAF-1 and for protein-DNA complex formation with Oct-1. Our findings indicate that VP16 contains additional sequences upstream of the acidic activation domain that may play a direct role in transactivation.