Specific and sensitive detection of Neisseria gonorrhoeae in clinical specimens by real-time PCR.

Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae in clinical samples. The target sequence is a 76-bp fragment of the 5' untranslated region of the opa genes that encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were negative in both the 16S rRNA test and the opa assay (confirming that the COBAS AMPLICOR test produces false positives), while 30% were positive in both assays. Three samples were opa positive and 16S rRNA negative, which may be due to the higher sensitivity of the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific, semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for confirmation of less specific tests.

A comparison of ligase chain reaction to polymerase chain reaction in the detection of Chlamydia trachomatis endocervical infections.

OBJECTIVE: To compare the reliability of ligase chain reaction (LCR) to polymerase chain reaction (PCR) in detecting Chlamydia trachomatis endocervical infections. METHODS: We conducted a prospective study of 486 patients at risk for chlamydial infection of the endocervix. We obtained two endocervical specimens from each patient and used LCR and PCR to detect C. trachomatis. Discrepant results between the two techniques were resolved by repeat testing and by testing for the major outer membrane protein (MOMP) gene, if necessary. We determined the sensitivity, specificity, positive predictive value, and negative predictive value for each test, using concordant results or MOMP gene results as the "gold standard". RESULTS: Of the 486 patients, 42 (8.6%) had evidence of C. trachomatis infection after resolution of discrepant results. Of the 42 true positive specimens, 41 were positive by initial LCR and 38 were positive by initial PCR. Of the 444 true negative specimens, none had a positive initial LCR result, while 2 had a positive initial PCR test. Therefore, compared to the gold standard, LCR had a sensitivity of 97.6% and specificity of 100%, while PCR had a sensitivity of 90% and a specificity of 99.5%. The positive and negative predictive values of LCR were 100% and 99.8%, respectively. PCR had a positive predictive value of 95% and a negative predictive value of 99.1%. The difference in sensitivity of LCR versus PCR was not statistically significant (P = .125). CONCLUSION: LCR and PCR perform equally well in detecting C. trachomatis endocervical infections.

Synchronization of estrus in yearling beef heifers with the melengestrol acetate/prostaglandin F(2alpha) system: efficiency of timed insemination 72 hours after prostaglandin treatment.

Two experiments were conducted to determine the conception rates of heifers time-inseminated following melengestrol acetate/prostaglandin F(2alpha) (MGA/PG) estrous synchronization treatment. In Experiment 1, timed insemination of heifers at 72 h after the PG injection, without regard for behavioral estrus, tended to improve (P < 0.15) the percentage of heifers pregnant to artificial insemination (AI) compared with that of synchronized heifers bred 12 h after they were first detected in estrus. In the timed-insemination treatment, heifers exhibiting behavioral estrus 48 to 72 h after PG tended to have an increased (P < 0.15) conception rate to AI compared with heifers exhibiting estrus within 48 h of PG administration. In Experiment 2, the number of heifers conceiving to AI following the MGA/PG estrous synchronization regimen was increased by mass insemination of all heifers not exhibiting estrus by 72 h after PG. The pregnancy rate to AI was higher in heifers with serum progesterone (P(4)) concentrations higher than 1 ng/ml compared with that of heifers with concentrations lower than 1 ng/ml. Of heifers with serum P(4) greater than 1 ng/ml, the pregnancy rate to AI tended to be higher when concentrations exceeded 2 ng/ml than when concentrations were 1 to 2 ng/ml. In cyclic heifers, timed insemination can increase the percentage of heifers pregnant after being synchronized with MGA/PG.

Application of the associated particle technique for the whole-body measurement of protein, fat and water by 14 MeV neutron activation analysis--a feasibility study.

A small and compact sealed tube neutron generator with an integral alpha particle detector has been used for applying the associated particle technique for prompt-gamma 14 MeV neutron activation analysis of total body carbon (TBC), total body nitrogen (TBN) and total body oxygen (TBO). Ground sheep meat samples in the weight range 20-40 kg and of varying composition have been scanned using two 12.5 cm diameter x 10 cm Nal(Tl) crystals for gamma-ray detection. The content of protein, fat and water was calculated from their fractional content of C, N and O using a four-compartment model of body composition, which included minerals. The precision for measuring TBC, TBN and TBO has been obtained from the mean count rates of ten repeat irradiations of the same sample. The accuracy has been confirmed by comparison against chemical analysis. The reproducibilities for measuring TBN have been found to be comparable to those obtained when the same samples were analysed using prompt-gamma thermal-neutron activation analysis in an existing body composition facility. Based on the results obtained, we conclude that an instrument comprising the neutron generator and four 15 cm x 15 cm x 45 cm NaI(Tl) gamma ray detectors can be assembled to determine, in vivo, protein, fat and water in an approximately 41 kg sample with precisions of 4.4%, 5.0% and 2.1% (CV) respectively within a 15 min scan. The radiation dose equivalent delivered due to neutrons would be approximately 0.03 mSv.

Whole body measurement of C, N and O using 14 MeV neutrons and the associated particle time-of-flight technique.

Our aim has been to construct a portable prototype instrument for measuring the whole body composition in vivo of growing lambs in terms of fat. protein and water by determining the mass of carbon, nitrogen and oxygen present. A small and compact sealed tube neutron generator which has the capability of exploiting the associated particle time-of-flight technique has been used for prompt gamma 14 MeV neutron activation analysis of C, N and O. This technique allows only gamma rays generated by neutron reactions within a defined volume to be recorded and offers a superior signal-to-noise ratio over existing prompt gamma neutron activation techniques. Based on the results obtained from irradiating a 41.4 kg meat phantom, we anticipate that an instrument comprising the neutron generator and four 15 x 15 x 45 cm Nal(TI) gamma ray detectors can be assembled to determine in vivo, protein, fat and water with precisions of 4.1, 5.4 and 1.2% (CV), respectively, within a 15 min scan. The radiation dose delivered would be ~0.03 mSv.

Performance, forage utilization, and ergovaline consumption by beef cows grazing endophyte fungus-infected tall fescue, endophyte fungus-free tall fescue, or orchardgrass pastures.

Two 120-d trials (May to September, 1988 and 1989) determined the effects of grazing tall fescue (two varieties) or orchardgrass on forage intake and performance by beef cows. Each summer, 48 cow-calf pairs grazed endophyte-infected Kentucky-31 tall fescue (KY-31), endophyte-free Mozark tall fescue (MOZARK), or Hallmark orchardgrass (OG) pastures (16 pairs/treatment). Forage OM intakes and digestibilities were determined during June and August each year. Cow and calf BW and milk production were determined every 28 d. During June of both years, OM intakes did not differ (P greater than .10) among treatments. During August of 1988, intakes were 18% lower (P less than .05) by KY-31 cows (1.6% of BW) than by MOZARK or OG cows (average 1.95% of BW); however, no differences (P greater than .10) were measured in August of 1989. Estimates of ergovaline consumption during June from KY-31 were between 4.2 (1988) and 6.0 mg/d (1989), whereas August estimates were between 1.1 (1988) and 2.8 mg/d (1989). Ergovaline in MOZARK estrusa was below detection limits, except in August of 1989. Cows that grazed KY-31 lost three times (P less than .01) more BW than cows that grazed MOZARK or OG (42 vs 9 and 13 kg, respectively). Milk production by KY-31 cows was 25% lower (P less than .01) than that by cows that grazed MOZARK or OG (6.0 vs average of 8.0 kg/d). Similarly, slower (P less than .01) calf gains were noted for KY-31 than for MOZARK or OG (.72 vs .89 and .88 kg/d, respectively). Cows grazing KY-31 experienced accelerated BW loss and reduced milk production and weaned lighter calves than did cows grazing MOZARK or OG. Decreased performance was not explained by consistently reduced forage intakes; hence, altered nutrient utilization was suspected.

Digestibility of vegetative and mature tall fescue greenchop with or without alfalfa greenchop substitution.

Nine ruminally and duodenally cannulated steers (average BW 355 kg) were used to evaluate effects of prebloom alfalfa greenchop substitution at 20% of DMI on utilization of late-May (high quality; HQ; Period 1) and mid-August (low quality; LQ; Period 2) tall fescue greenchop. Alfalfa inclusion did not influence (P greater than .10) diet ad libitum DMI during Period 1 but it decreased (P less than .10) DMI during Period 2. Ruminal and total tract DM, cell wall, and GE digestibility of HQ were unaffected (P greater than .10) by alfalfa inclusion; however, digestibility of these constituents in LQ was increased (P less than .03) by alfalfa substitution. Alfalfa substitution did not influence (P greater than .10) dietary cell wall monosaccharide disappearance. Ruminal CP digestibility was greater (P less than .10) when steers received alfalfa, but microbial efficiency (grams of bacterial N/kilogram of OM truly digested in the rumen) was not enhanced (P greater than .10) by alfalfa inclusion in either HQ or LQ diets. There was a trend (P = .15) for greater microbial efficiency with alfalfa substitution to LQ. Ruminal particulate passage rate did not differ (P greater than .10) between treatments for either stage of maturity. Fluid passage rate was faster (P less than .10) in steers that received only LQ (7.1%/h) than in those fed LQ substituted with 20% alfalfa (5.0 %/h). Our data suggest that alfalfa inclusion in a low-quality fescue diet enhanced cell wall and GE digestibility.

The human lysozyme gene. Sequence organization and chromosomal localization.

We have isolated two overlapping recombinant lambda-phage clones from a genomic lambda-EMBL3 library containing 25 kb of the human lysozyme gene region. Furthermore a full-lenght human lysozyme cDNA clone of 1.5 kb was isolated from a human placenta cDNA library. Nucleotide sequences of the entire structural gene and the cDNA clone were determined. The human lysozyme gene spans 5856 bp and its sequence organization with four exons and three introns is homologous to the chicken lysozyme gene and the human alpha-lactalbumin gene. Human and chicken lysozyme genes differ mainly in the size of their introns and 3' non-coding region. Four Alu repetitive elements were found in the human lysozyme gene, one in each intron and one on the fourth exon. Lysozyme transcripts of 1.6 kb and 0.6 kb in size were detected in human myeloid cell lines U-937, HL-60 and THP-1 and surprisingly in human hepatoma cell lines HepG2 and Hep3B. The lysozyme gene locus was assigned to human chromosome 12 by hybridization to a panel of DNAs from human-rodent somatic cell hybrids.

Rat antibodies as probes for the characterization of progesterone receptor A and B proteins from laying hen oviduct cytosol.

The chicken oviduct contains two different hormone binding forms of the progesterone receptor, A and B. We have prepared rat antisera against both forms of the receptor partially purified from laying hen oviduct. The anti-progesterone receptor A antiserum reacts with both receptor forms on Western blots, while the anti-progesterone receptor B antiserum reacts mainly with the B form. Both antisera also react with the native progesterone receptor proteins as shown by sedimentation analysis of the antibody-receptor complexes. Receptors A and B are recognized on Western blots of total protein from dissolved tissue, indicating that both forms are likely to be physiological components. Epitope mapping experiments show that immunogenicity of both receptor molecules is restricted to structurally related protein domains of 28 kDa in receptor A and of 52 kDa in receptor B.

Drosophila and vertebrate myb proteins share two conserved regions, one of which functions as a DNA-binding domain.

We report the nucleotide sequence of a cDNA clone of the Drosophila melanogaster homologue of c-myb, a member of the class of vertebrate transforming genes encoding nuclear proteins. We predict the mol. wt of the Drosophila myb (D-myb) protein to be 74,000. The D-myb protein contains two clusters of sequences homologous to vertebrate myb proteins, surrounded by sequences lacking homology. These results extend previous evidence for the existence of a D. melanogaster homologue of c-myb and identify two highly conserved and therefore presumably functionally important domains of c-myb proteins. DNA-binding experiments indicate that the NH2-proximal of the two homology regions functions as a DNA-binding domain. Based on the absence of the COOH-proximal homology region in truncated oncogenic derivatives of c-myb it is likely that this homology region encodes a function whose loss is involved in activating the oncogenic potential of c-myb.

Nevus leiomyomatosus systematicus.

Leiomyomas of the skin are classified as pilar, genital, or angioleiomyoma depending on the site of origin. Pilar leiomyomas are the most common form, and usually multiple tumors are found in a single patient. An unusual variant of multiple pilar leiomyoma is nevus leiomyomatosus systematicus, a descriptive term used when tumors are wide-spread and patterned, suggesting a nevoid condition. Many of the large pilar tumors are painful and require surgical excision for relief of symptoms.

High-Resolution Stark Spectroscopy in the nu(4) Band of NH(3) Using a Thin-Film Diode Laser.

Thin-film PbTe diode lasers are used for Doppler-limited Stark spectroscopy on the nu(4) vibration-rotation band of NH(3) in the 1545-1595-cm(-1) region. The lasers operate cw below 20 K and are frequency tuned by varying the diode current. The nu(4) excited state dipole moment of NH(3) is found to be about 1% less than the ground state value.