Senna is a pod or leaf of Senna alexandrina P. Mill and is used as a stimulant laxative. In the large intestine, bacterial enzymes break sennosides and release rhein-9-anthrone, the active form for the laxative effect. To determine potential toxic effects of senna, a 5-week dose range finding study in the C57BL/6N mouse and a 40-week toxicology and carcinogenesis study in the C3B6.129F1-Trp53tm1Brd N12 haploinsufficient (p53+/−) mouse were conducted. In the 5-week study, C57BL/6N mice were exposed up to 10,000 ppm senna in feed. Increased incidences of epithelial hyperplasia of the cecum and colon were observed in males and females exposed to 5,000 or 10,000 ppm senna. These intestinal lesions were not considered to be of sufficient severity to cause mortality and, thus, in the p53+/− mouse 40-week study, the high dose of 10,000 ppm was selected. Significant increases in the incidences of epithelial hyperplasia of the colon and cecum were observed at 10,000 ppm in p53(+/−) males and females, and the incidence of hyperplasia of the colon was significantly increased at 3,000 ppm in females. In conclusion, the large intestine was the major target of senna-induced toxicity in both wild-type and the p53+/− mouse model. There was no neoplastic change, when senna was administered to p53 +/− mouse.
Senna; Trp53; carcinogenesis; toxicity; laxative; large intestine
Despite the ubiquity of chemoautotrophic symbioses at hydrothermal vents, our understanding of the influence of environmental chemistry on symbiont metabolism is limited. Transcriptomic analyses are useful for linking physiological poise to environmental conditions, but recovering samples from the deep sea is challenging, as the long recovery times can change expression profiles before preservation. Here, we present a novel, in situ RNA sampling and preservation device, which we used to compare the symbiont metatranscriptomes associated with Alviniconcha, a genus of vent snail, in which specific host–symbiont combinations are predictably distributed across a regional geochemical gradient. Metatranscriptomes of these symbionts reveal key differences in energy and nitrogen metabolism relating to both environmental chemistry (that is, the relative expression of genes) and symbiont phylogeny (that is, the specific pathways employed). Unexpectedly, dramatic differences in expression of transposases and flagellar genes suggest that different symbiont types may also have distinct life histories. These data further our understanding of these symbionts' metabolic capabilities and their expression in situ, and suggest an important role for symbionts in mediating their hosts' interaction with regional-scale differences in geochemistry.
symbiosis; hydrothermal vents; metatranscriptomics; chemoautotrophy; Alviniconcha
Despite significant heritability of autism spectrum disorders (ASDs), their extreme genetic heterogeneity has proven challenging for gene discovery. Studies of primarily simplex families have implicated de novo copy number changes and point mutations, but are not optimally designed to identify inherited risk alleles. We apply whole exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, POMGNT1), some implicated for the first time in ASD. At least some of these genes show biallelic mutations in nonconsanguineous families as well. These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated. Our study shows the utility of WES for identifying specific genetic conditions not clinically suspected and the importance of partial loss of gene function in ASDs.
The fabrication of biomimetic scaffolds is a critical component to fulfill the promise of functional tissue engineered materials. We describe herein a simple technique, based on printed circuit board manufacturing, to produce novel templates for electrospinning scaffolds for tissue engineering applications. This technique facilitates fabrication of electrospun scaffolds with templated architecture, which we defined as a scaffold's bulk mechanical properties being driven by its fiber architecture. Electrospun scaffolds with templated architectures were characterized with regard to fiber alignment and mechanical properties. Fast Fourier transform analysis revealed a high degree of fiber alignment along the conducting traces of the templates. Mechanical testing showed that scaffolds demonstrated tunable mechanical properties as a function of templated architecture. Fibroblast seeded scaffolds were subjected to a peak strain of 3% or 10% at 0.5 Hz for 1 hour. Exposing seeded scaffolds to the low strain magnitude (3%) significantly increased collagen I gene expression compared to the high strain magnitude (10%) in a scaffold architecture dependent manner. These experiments indicate that scaffolds with templated architectures can be produced and modulation of gene expression is possible with templated architectures. This technology holds promise for the long term goal of creating tissue engineered replacements with the biomechanical and biochemical make-up of native tissues.
electrospinning; tissue engineering; anisotropic scaffold; collagen I; cyclic strain
hypoparathyroidism; calcium; epidemiology; genetics; parathyroid hormone; hypocalcemia
p21-activated kinase (PAK) has been implicated in the inflammatory activation of endothelial cells by disturbed fluid shear stress, which is the initiating stimulus in atherosclerosis. The study addresses whether PAK1 contributes to inflammatory marker expression in endothelial cells at atherosclerosis-susceptible regions of arteries in vivo.
Aortas from WT and PAK1-/- C57BL/6J mice on a normal chow diet were fixed, dissected and processed for immunohistochemistry using a panel of inflammatory markers. We visualized and quantified staining in the endothelium at the greater and lesser curvatures of the arch of aorta, as atherosclerosis-resistant and susceptible regions, respectively.
Fibronectin, VCAM-1 and the activated RelA NF-κB subunit were localized to the lesser curvature and decreased in PAK1-/- mice. The activated RelB NF-κB subunit was also localized to the lesser curvature but was increased in PAK1-/- mice. Low levels of staining for ICAM-1 and the monocyte/macrophage marker Mac2 indicated that overall inflammation in this tissue was minimal.
These data show that PAK1 has a significant pro-inflammatory function at atherosclerosis-prone sites in vivo. These effects are seen in young mice with very low levels of inflammation, suggesting that inflammatory activation of the endothelium is primarily biomechanical. Activation involves NF-κB, expression of leukocyte recruitment receptors and fibronectin deposition. These results support and extend in vitro studies demonstrating that PAK contributes to activation of inflammatory pathways in endothelial cells by fluid shear stress.
Fluid shear stress; Endothelial cells; Vascular inflammation; Fibronectin
Unrelated donor (URD) BMT is an effective treatment for leukemia in children, but success is limited by graft versus host disease (GVHD) and relapse. In this study we describe the incidence and risk factors for GVHD over time in children receiving URD BMT. We analyzed outcomes of 638 myeloablative URD BMT performed between 1990 and 2003 for treatment of AML, ALL, CML or MDS, using the Center for International Blood & Marrow Transplant Research (CIBMTR) database. Recipients were <18 years of age, and had available high resolution HLA typing for HLA A, B, C and DRB1. Overall, 27% of recipients developed aGVHD grades III-IV and risk was significantly higher in recipients of T-replete compared with T-depleted grafts (OR 3.12, 95%CI 2.02-4.83; p< 0.0001). Acute GVHD significantly reduced risk of relapse in children with ALL (OR 0.34 95% CI 0.13-0.86, p=0.0052) but not AML (OR 0.58, 95% CI 0.22-2.98; p=0.26). Risk of aGVHD was higher in children transplanted in earlier years (1990-1998, n=365) compared to 1999-2003 (OR 1.93 95%CI 1.27-2.91; p=0.002). We conclude that outcomes have changed significantly over time, with reduced risk of aGVHD in more recent transplants.
The correlation between dehydroepiandrosterone sulfate (DHEAS) decline and age led to the hypothesis that DHEAS might be a marker of primary aging, though conflicting data from observational studies of mortality do not support this. We evaluated concurrent DHEAS and functional decline in a very old cohort to test if DHEAS change tracks with functional change during aging.
DHEAS and functional performance (gait speed, grip strength, Modified Mini-Mental State Examination [3MSE] score, and digit symbol substitution test [DSST] score) were measured in 1996–1997 and 2005–2006 in 989 participants in the Cardiovascular Health Study All Stars study (mean age 85.2 years in 2005–2006, 63.5% women and 16.5% African American). We used multivariable linear regression to test the association of DHEAS decline with functional decline.
After adjustment, each standard deviation decrease in DHEAS was associated with greater declines in gait speed (0.12 m/s, p = .01), grip strength (0.09 kg, p = .03), 3MSE score (0.13 points, p < .001), and DSST score (0.14 points, p = .001) in women only. Additional adjustment for baseline DHEAS attenuated the association with grip strength but did not alter other estimates appreciably, and baseline DHEAS was unassociated with functional decline.
In this cohort of very old individuals, DHEAS decline tracked with declines in gait speed, 3MSE score, and DSST score, but not grip strength, in women independent of baseline DHEAS level. DHEAS decline might be a marker for age-associated performance decline, but its relevance is specific to women.
Aging; Biomarker; Dehydroepiandrosterone sulfate; Function
Cresols, monomethyl derivatives of phenol, are high production chemicals with potential for human exposure. The three isomeric forms of cresol are used individually or in mixtures as disinfectants, preservatives, and solvents or as intermediates in the production of antioxidants, fragrances, herbicides, insecticides, dyes, and explosives. Carcinogenesis studies were conducted in groups of 50 male F344/N rats and 50 female B6C3F1 mice exposed to a 60:40 mixture of m and p cresols (m-/p-cresol) in feed. Rats and mice were fed diets containing 0, 1500, 5000, or 15,000 ppm and 0, 1000, 3000, or 10,000 ppm, respectively. Survival of each exposed group was similar to that of their respective control group. Mean body weight gains were depressed in rats exposed to 15,000 ppm and in mice exposed to 3000 ppm and higher. A decrease of 25% over that of controls for the final mean body weight in mice exposed to 10,000 ppm appeared to be associated with lack of palatability of the feed. A marginally increased incidence of renal tubule adenoma was observed in the 15,000 ppm-exposed rats. The increased incidence was not statistically significant, but did exceed the range of historical controls. No increased incidence of hyperplasia of the renal tubules was observed; however, a significantly increased incidence of hyperplasia of the transitional epithelium associated with an increased incidence of nephropathy was observed at the high exposure concentration. The only significantly increased incidence of a neoplastic lesion related to cresol exposure observed in these studies was that of squamous cell papilloma in the forestomach of 10,000 ppm-exposed mice. A definitive association with irritation at the site-of-contact could not be made because of limited evidence of injury to the gastric mucosa at the time of necropsy. However, given the minimal chemical-related neoplastic response in these studies, it was concluded that there was no clear evidence of carcinogenicity in male rats or female mice exposed to the cresol mixture.
carcinogenicity; cresols; m-cresol; p-cresol; forestomach papilloma; renal tubule adenoma
The disposition of the 14C-labeled polybrominated diphenyl ether (PBDE), 2,2′,4,4′,5,5′-hexaBDE (BDE153) was investigated in rodents following single and multiple doses and in a mixture with radiolabeled 2,2′,4,4′-tetraBDE (BDE47) and 2,2′,4,4′,5-pentaBDE (BDE99).In single exposure studies, there was little or no effect of dose on BDE153 disposition in male rats in the range of 1–100 µmol/kg. No major sex or species differences in the in vivo fate of BDE153 were detected. BDE153 was: 1) approximately 70% absorbed in rats or mice following gavage. 2) retained in tissues. 3) poorly metabolized and slowly excreted.Mixture studies indicated that, relative to each other, more BDE47 was distributed to adipose tissue, more BDE153 accumulated in liver, and BDE99 was metabolized to the greatest extent. BDE153 was probably retained in liver due to minimal metabolism and elimination after "first pass" distribution to the tissue following gavage.
Polybrominated diphenyl ethers; PBDE; metabolism and disposition; persistent organic pollutants
To evaluate whether T cell activation, as reflected by levels of soluble interleukin 2 receptor (sIL2R), soluble CD30 (sCD30), IL‐10 and B cell activator of the tumour necrosis factor family (BAFF) at diagnosis and during initial follow‐up, is predictive for persistent or renewed antineutrophil cytoplasmic antibody (ANCA) positivity and clinical relapse in patients with vasculitis associated with proteinase 3‐antineutrophil cytoplasmic antibodies (PR3‐ANCA).
87 Patients with PR3‐ANCA‐associated vasculitis and at least 2 years of follow‐up were included in the study. At diagnosis, and at 3, 6, 12, 18 and 24 months after diagnosis, cytoplasmic ANCA titres were detected by indirect immunofluorescence (IIF), and PR3‐ANCA, sIL2R, sCD30, IL‐10 and BAFF levels were assessed by ELISA. 31 healthy volunteers provided plasma samples for comparison. Levels of immune markers were related to ANCA positivity and relapse during follow‐up.
Plasma levels of sIL2R, sCD30 and BAFF were higher in patients than in controls at all time points. Plasma levels of sIL2R, sCD30 and IL‐10 were higher at diagnosis and relapse than during remission. At 18 months, sCD30 (p<0.001) and sIL2R levels (p = 0.01) were significantly higher in PR3‐ANCA‐positive patients (detected by ELISA) than in PR3‐ANCA‐negative patients. ANCA‐positive patients detected by ELISA or IIF at 24 months had significantly higher plasma sCD30 levels (p = 0.02 and p = 0.03, respectively) than ANCA‐negative patients.
Increased T cell activation in patients with ANCA‐associated vasculitis in remission during and after immunosuppressive treatment is associated with persistent or renewed ANCA positivity.
Hospital surveillance was established in the Nile River Delta to increase the understanding of the epidemiology of diarrheal disease among Egyptian children. Between September 2000 and August 2003, samples obtained from children less than 5 years of age who had diarrhea and who were seeking hospital care were cultured for enteric bacteria. Colonies from each culture with a morphology typical of that of Escherichia coli were tested for the heat-labile (LT) and heat-stable (ST) toxins by a GM-1-specific enzyme-linked immunosorbent assay and colonization factor (CF) antigens by an immunodot blot assay. Enterotoxigenic E. coli (ETEC) isolates were recovered from 320/1,540 (20.7%) children, and ETEC isolates expressing a known CF were identified in 151/320 (47%) samples. ST CFA/I, ST CS6, ST CS14, and LT and ST CS5 plus CS6 represented 75% of the CFs expressed by ETEC isolates expressing a detectable CF. Year-to-year variability in the proportion of ETEC isolates that expressed a detectable CF was observed (e.g., the proportion that expressed CFA/I ranged from 10% in year 1 to 21% in year 3); however, the relative proportions of ETEC isolates expressing a CF were similar over the reporting period. The proportion of CF-positive ETEC isolates was higher among isolates that expressed ST. ETEC isolates expressing CS6 were isolated significantly less often (P < 0.001) than isolates expressing CFA/I in children less than 1 year of age. Macrorestriction profiling of CFA/I-expressing ETEC isolates by using the restriction enzyme XbaI and pulsed-field gel electrophoresis demonstrated a wide genetic diversity among the isolates that did not directly correlate with the virulence of the pathogen. The genome plasticity demonstrated in the ETEC isolates collected in this work suggests an additional challenge to the development of a globally effective vaccine for ETEC.
Military personnel with traveler's diarrhea (n = 202) while deployed to Incirlik Air Base, Turkey, from June to September 2002 were evaluated for pathogen-specific immune responses. Serologic and fecal immunoglobulin A (IgA) titers to enterotoxigenic Escherichia coli antigens (CS6, CS3, and LT) were quite low. In contrast, subjects with Campylobacter infections had high serologic and fecal IgA responses.
Background: Several autoimmune disorders are complicated by excess cardiovascular disease. In addition to traditional risk factors, non-traditional risk factors such as endothelial activation and excessive vascular remodelling might be determinants of the progression of atherosclerosis in patients with an autoimmune disease.
Objective: To evaluate whether patients with Wegener's granulomatosis (WG) have an increased prevalence of atherosclerosis and to determine predisposing factors.
Methods: 29 WG patients (19 men; mean (SD) age, 53 (14) years) with inactive disease and 26 controls (16 men; age 53 (15) years) were studied. Common carotid intima–media thickness (IMT) was measured by ultrasound. In all individuals traditional risk factors for cardiovascular disease were determined. High sensitivity C reactive protein (hsCRP) was measured. Endothelial activation was assessed by measuring thrombomodulin, vascular cell adhesion molecule-1, and von Willebrand factor. As a marker of vascular remodelling matrix metalloproteinases (MMP-3 and MMP-9) and TIMP-1 were measured.
Results: IMT was increased in WG patients compared with controls (p<0.05). No differences in traditional risk factors and endothelial activation markers between patients and controls were found. Levels of hsCRP, MMPs, and TIMP-1 were increased in WG patients (p<0.05).
Conclusions: Increased IMT found in WG patients cannot be explained by an increased prevalence of traditional risk factors. Although endothelial activation markers in WG patients with inactive disease were not increased, the raised levels of hsCRP, MMPs, and TIMP-1 suggest that enhanced inflammation and excessive vascular remodelling are contributing factors in the development of accelerated atherosclerosis in WG.
Background: Angiotensin 1 converting enzyme (ACE) inhibitors reduce morbidity and mortality after coronary artery bypass graft surgery (CABG). This benefit may result from an anti-inflammatory action.
Objective: To examine the effect of ACE inhibition on interleukin 6 (IL-6) concentrations after CABG.
Patients and methods: 161 patients undergoing elective first time CABG were recruited, of whom 41 (25%) were receiving ACE inhibitor treatment; 21 patients with confounding postoperative complications were excluded. After these exclusions there were 33 patients (24%) on ACE inhibitor treatment. Plasma IL-6 was measured preoperatively and again six hours after CABG.
Results: Baseline IL-6 concentrations (geometric mean (SEM)) were non-significantly lower among the patients receiving ACE inhibitors (3.7 (0.1) v 4.3 (0.1) pg/ml, p = 0.12). Overall, post-CABG IL-6 concentrations increased significantly (mean rise 177 (12) pg/ml, p < 0.0005). This response was blunted among ACE inhibitor treated patients. Median increases in IL-6 concentrations were 117 v 193 pg/ml, for treated v non-treated patients, respectively (Kruskal–Wallis, p = 0.02), with peak postoperative IL-6 concentrations lower among the subjects receiving ACE inhibitors than in untreated subjects (142 (19) v 196 (13) pg/ml, p = 0.02). The effect of ACE inhibitors remained significant after multivariate analysis (p = 0.018).
Conclusions: ACE inhibitor treatment is associated with a reduction in IL-6 response to CABG. The data suggest that this class of drug may have a direct anti-inflammatory effect, which could explain some of its clinical benefit.
angiotensin converting enzyme; inflammation; interleukin 6; coronary artery bypass graft
A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells.
In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.
This paper reports a number of cases of patients attending an accident and emergency (A&E) department claiming to be HIV positive when they have been tested negative and are known to be negative by other departments in the hospital. The reasons for these patients claims are not always apparent. These patients may place an inappropriate workload on an already busy department. We caution doctors working in A&E departments to be vigilant when dealing with patients who claim to be HIV positive when there are no clinical or laboratory findings to substantiate the claim and we recommend liaison between relevant departments within a hospital and the patient's general practitioner (GP) when dealing with these patients.
Haemophilus influenzae has an absolute requirement for heme for aerobic growth. This organism can satisfy this requirement by synthesizing heme from iron and protoporphyrin IX (PPIX). H. influenzae type b (Hib) strain DL42 was found to be unable to form single colonies when grown on a medium containing free iron and PPIX in place of heme. In contrast, the nontypeable H. influenzae (NTHI) strain TN106 grew readily on the same medium. A genomic library from NTHI strain TN106 was used to transform Hib strain DL42, and recombinants were selected on a medium containing iron and PPIX in place of heme. A recombinant plasmid with an 11.5-kb NTHI DNA insert was shown to confer on Hib strain DL42 the ability to grow on iron and PPIX. Nucleotide sequence analysis revealed that this NTHI DNA insert contained three genes, designated hitA, hitB, and hitC, which encoded products similar to the SfuABC proteins of Serratia marcescens, which have been shown to constitute a periplasmic binding protein-dependent iron transport system in this enteric organism. The NTHI HitA protein also was 69% identical to the ferric-binding protein of Neisseria gonorrhoeae. Inactivation of the cloned NTHI hitC gene by insertion of an antibiotic resistance cartridge eliminated the ability of the recombinant plasmid to complement the growth deficiency of Hib DL42. Construction of an isogenic NTHI TN106 mutant lacking a functional hitC gene revealed that this mutation prevented this strain from growing on a medium containing iron and PPIX in place of heme. This NTHI hitC mutant was also unable to utilize either iron bound to transferrin or iron chelates. These results suggest that the products encoded by the hitABC genes are essential for the utilization of iron by NTHI.
Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.
In a study of 1,486 men attending two sexually transmitted disease clinics, of whom 891 had no symptoms of urethritis, we compared an enzyme immunoassay (EIA) (Baxter-Bartels, formerly Northumbria AntigEnz) of urine sediment to urethral culture for the detection of Chlamydia trachomatis. C. trachomatis prevalence by culture alone was 7.7% in asymptomatic men and 10.9% in symptomatic men. Discrepant results between EIA of urine and urethral culture were evaluated by direct fluorescent-antibody staining (DFA) for elementary bodies in urine sediment or in culture transport media. When chlamydial infection was defined as either a positive urethral culture or positive EIA confirmed by DFA, chlamydia prevalence increased to 8.9% in asymptomatic men and 11.6% in symptomatic men. The urine EIA sensitivity, specificity, and positive and negative predictive values for chlamydia detection in asymptomatic men were 84.8, 99.3, 91.8, and 98.5%, respectively, with nearly identical results for symptomatic men. The sensitivities of urethral culture alone compared with the combination of urethral culture and urine EIA (with DFA confirmation) were 87.3 and 94.3% for asymptomatic and symptomatic men, respectively. The present EIA of urine sediment is both highly sensitive and specific for the detection of C. trachomatis in asymptomatic men, thus providing a noninvasive screening method for chlamydia infection in asymptomatic men attending sexually transmitted disease clinics.
The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.
Many human colonic Bacteroides strains carry large (greater than 70-kbp) self-transmissible chromosomal tetracycline resistance (Tcr) elements. These Tcr elements can also mediate the excision and circularization of discrete nonadjacent segments of chromosomal DNA which are designated NBUs (nonreplicating Bacteroides units). We have localized a 6.5-kbp segment of Tcr element DNA that mediates NBU excision and circularization. Analysis of the DNA sequence of this region indicated that it contained three open reading frames, all transcribed in the same direction. The first gene was the Tcr gene, tetQ. The second two open reading frames exhibited amino acid similarity to known two-component regulatory systems. Complementation and gene fusion data supported the hypothesis that the three genes were organized in an operon. Transcription from the tetQ promoter region was inducible by tetracycline, as might be expected from the previous finding that NBU excision was detectable only in cells preexposed to tetracycline. The 6.5-kbp region appeared to be essential not only for NBU excision but also for self-transfer of the elements, another activity that is enhanced by preexposure to tetracycline. Accordingly, the two genes downstream of tetQ have been designated rteA and rteB (regulation of Tcr elements).
Membrane bound and soluble forms of a high-affinity folate binding protein have been found in kidney, placenta, serum, milk, and in several cell lines. The two forms have similar binding characteristics for folates, are immunologically cross-reactive and based upon limited amino acid sequence data, are nearly identical. Based upon pulse-chase experiments, a precursor-product relationship has been suggested. The membrane form has been shown to mediate the transport of folate in cells grown in physiological concentrations of folate. A function for the soluble form has not yet been identified. We constructed a cDNA library from a human carcinoma cell line, Caco-2, which expresses the membrane form abundantly. The library was screened and a near full-length cDNA for the folate binder was isolated. Transfection of COS cells with the cDNA inserted in an expression vector resulted in marked overexpression of a membrane-associated folate binder as assessed by direct binding of radiolabeled folate and by indirect immunofluorescence. The deduced amino acid sequence is not consistent with a typical membrane spanning domain but rather with a signal for anchoring via a glycosyl-phosphatidylinositol linkage. Release of the binder with a phosphatidylinositol-specific phospholipase C strongly supports this hypothesis.