Defenses against oxidative stress in Neisseria gonorrhoeae: A system tailored for a challenging environment

Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 muM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.

Large mitochondrial repeats multiplied during the polymerase chain reaction

The number of repeats in repetitive DNA like micro- and minisatellites is often determined by polymerase chain reaction (PCR). When we counted repeats in an array of mitochondrial repeats in the cattle tick (Boophilus microplus) we found that the number of repeats increased during PCR. Multiplication of the repeats was independent of the primers used to amplify the region, the PCR annealing temperature and the length of the PCR product. The use of PCR to determine the number of repeats in arrays needs to be reassessed. For long repeats, a subset of samples should always be analysed by Southern blot hybridization to confirm the PCR results.

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gem in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen In a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction In the alcohol-affected brain.

Differential expression of mitochondrial NADH dehydrogenase in ethanol-treated rat brain: Revealed by differential display

The technique of polymerase chain reaction (PCR) differential display was used to detect alterations in gene expression after chronic alcohol administration. Male Wistar rats were treated with ethanol vapor for 14 days. The cDNA generated from mRNA isolated from the hippocampi of ethanol-treated and control animals was compared by PCR differential display. A differentially expressed cDNA fragment was used to screen mRNA samples by Northern analysis. The level of a mRNA was significantly elevated (x 2.5) in the hippocampus, but not the cortex of alcohol-treated rats up to 48 hr after withdrawal. Sequence analysis of the cDNA fragment revealed an almost perfect homology to rat mitochondrial NADH dehydrogenase subunit 4 mRNA. The selective induction of this mRNA in alcohol-treated rat brain areas suggests altered metabolic processes and possible dysfunction of the mitochondria. The technique of PCR differential display may prove useful in further analysis of gene expression during alcohol dependence and withdrawal.

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV 2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR) = 2.67, 95% CI = 1.59-4.47, p = 0.0171]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV 2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.

A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples

Cell culture and direct fluorescent antibody (DFA) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections. Multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) is a sensitive, specific, and rapid method for detecting several DNIA and RNA viruses in a single specimen. We developed a m-RT-PCR assay that utilizes multiple virus-specific primer pairs in a single reaction mix combined with an enzyme-linked amplicon hybridization assay (ELAHA) using virus-specific probes targeting unique gene sequences for each virus. Using this m-RT-PCR-ELAHA, we examined the presence of seven respiratory viruses in 598 nasopharyngeal aspirate (NPA) samples from patients with suspected respiratory infection. The specificity of each assay was 100%. The sensitivity of the DFA was 79.7% and the combined DFA/culture amplified-DFA (CA-DFA) was 88.6% when compared to the m-RT-PCR-ELAHA. Of the 598 NPA specimens screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADM), 2% for influenza A (Flu A) virus, 0.3% for influenza B (Flu B) virus, 1% for parainfluenza type I virus (PIV1), 1% for parainfluenza type 2 virus (PIV2), 5.5% for parainfluenza type 3 virus (PIV3), and 21% for respiratory syncytial virus (RSV). The enhanced sensitivity, specificity, rapid result turnaround time and reduced expense of the m-RT-PCR-ELAHA compared to DFA and CA-DFA, suggests that this assay would be a significant improvement over traditional assays for the detection of respiratory viruses in a clinical laboratory.

Detection and differentiation of Plasmodium species by polymerase chain reaction and colorimetric detection in blood samples of patients with suspected malaria

Polymerase chain reaction (PCR) is now recognized as a sensitive and specific method for detecting Plasmodium species in blood. In this Study. we tested 279 blood samples, from patients with Suspected malaria, by a PCR assay utilizing species-specific colorimetric detection. and compared the results to light microscopy. Overall, both assays were in agreement for 270 of the 279 specimens. P. vivax was detected in 131 (47.0%) specimens. P. falciparum in 64 (22.9%) specimens, P. ovale in 6 (2.1%) specimens, and P. malariae in 5 (1.8%) specimens. Both P. falciparum and P. vivax were detected in a further 10 (3.6%) specimens, and 54 (19.3%) specimens were negative by both assays. In the remaining nine specimens, microscopy either failed to detect the parasite or incorrectly identified the species present. In summary, the sensitivity, specificity and simplicity of the PCR assay makes it particularly suitable for use in a diagnostic laboratory. (C) 2004 Elsevier Inc. All rights reserved.

Phylogeny and biogeography of the freshwater crayfish Euastacus (Decapoda : Parastacidae) based on nuclear and mitochondrial DNA

Euastacus crayfish are endemic to freshwater ecosystems of the eastern coast of Australia. While recent evolutionary studies have focused on a few of these species, here we provide a comprehensive phylogenetic estimate of relationships among the species within the genus. We sequenced three mitochondrial gene regions (COI, 16S, and 12S) and one nuclear region (28S) from 40 species of the genus Euastacus, as well as one undescribed species. Using these data, we estimated the phylogenetic relationships within the genus using maximum-likelihood, parsimony, and Bayesian Markov Chain Monte Carlo analyses. Using Bayes factors to test different model hypotheses, we found that the best phylogeny supports monophyletic groupings of all but two recognized species and suggests a widespread ancestor that diverged by vicariance. We also show that Eitastacus and Astacopsis are most likely monophyletic sister genera. We use the resulting phylogeny as a framework to test biogeographic hypotheses relating to the diversification of the genus. (c) 2005 Elsevier Inc. All rights reserved.

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n=140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n=18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.

Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus

A 13-residue peptide sequence from a respiratory syncitial virus fusion protein was constrained in an alpha-helical conformation by fusing two back-to-back cyclic alpha-turn mimetics. The resulting peptide, Ac-(3 -> 7; 8 -> 12)-bicyclo-FP[KDEFD][KSIRD]V-NH2, was highly alpha-helical in water by CD and NMR spectroscopy, correctly positioning crucial binding residues (F488, I491, V493) on one face of the helix and side chain-side chain linkers on a noninteracting face of the helix. This compound displayed potent activity in both a recombinant fusion assay and an RSV antiviral assay (IC50 = 36 nM) and demonstrates for the first time that back-to-back modular alpha-helix mimetics can produce functional antagonists of important protein-protein interactions.

Determination of organ tropism of Newcastle disease virus (strain I-2) by virus isolation and reverse transcription-polymerase chain reaction

The vaccines 1-2 and V4 are avirulent strains of Newcastle disease virus. Organ tropism of strain V4 has been determined and the virus has a predilection for the digestive tract. Tropism of strain 1-2 has not yet been determined. The objective of this study was to determine the distribution of strain 1-2 in various body organs and fluids following vaccination in comparison with V4. Four-week-old chickens were vaccinated by eye drop separately with these two avirulent strains. Virus isolation and the reverse transcription-polymerase chain reaction technique were employed to detect 1-2 and V4 viruses in various tissues and body fluids for 7 days following vaccination. Tissues from the respiratory tract showed earlier positive signals than tissues from other organs for chickens vaccinated with strain 1-2. Conversely, tissues from mainly digestive tract produced earlier positive signals than from respiratory tract and other organs from chickens vaccinated with strain V4. In early infection, strain 1-2 had preferential predilection for the respiratory tract and strain V4 for the digestive tract. Later after vaccination, other organs showed positive results from chickens vaccinated with both 1-2 and V4 strains. The differences in organ tropism observed in this study suggest that 1-2 may perform better than V4 as a live vaccine strain.

Respiratory and cardiovascular responses to hypoxia in the Australian lungfish

Simultaneous measurements of pulmonary blood flow (qPA), coeliacomesenteric blood flow (qCoA), dorsal aortic blood pressure (PDA), heart rate (fH) and branchial ventilation frequency (fv) were made in the Australian lungfish, /Neoceratodus forsteri, /during air breathing and aquatic hypoxia. The cho­linergic and adrenergic influences on the cardiovascular system were investigated during normoxia using pharmacological agents, and the presence of catecholamines and serotonin in different tissues was investi­gated using histochemistry. Air breathing rarely occurred during normoxia but when it did, it was always associated with increased pulmonary blood flow. The pulmonary vasculature is influenced by both a cho­linergic and adrenergic tonus whereas the coeliacomesenteric vasculature is influenced by a β-adrenergic vasodilator mechanism. No adrenergic nerve fibers could be demonstrated in /Neoceratodus /but catecholamine-containing endothelial cells were found in the atrium of the heart. In addition, serotonin-­immunoreactive cells were demonstrated in the pulmonary epithelium. The most prominent response to aquatic hypoxia was an increase in gill breathing frequency followed by an increased number of air breaths together with increased pulmonary blood flow. It is clear from the present investigation that /Neoceratodus /is able to match cardiovascular performance to meet the changes in respiration during hypoxia.

Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity following moderate and high intensity exercise

Intense exercise stimulates the systemic release of a variety of factors that alter neutrophil surface receptor expression and functional activity. These alterations may influence resistance to infection after intense exercise. The aim of this study was to examine the influence of exercise intensity on neutrophil receptor expression, degranulation (measured by plasma and intracellular myeloperoxidase concentrations), and respiratory burst activity. Ten well-trained male runners ran on a treadmill for 60 min at 60% [moderate-intensity exercise (MI)] and 85% maximal oxygen consumption [high-intensity exercise (HI)]. Blood was drawn immediately before and after exercise and at 1 h postexercise. Immediately after HI, the expression of the neutrophil receptor CD16 was significantly below preexercise values (P < 0.01), whereas MI significantly reduced CD35 expression below preexercise values (P < 0.05). One hour after exercise at both intensities, there was a significant decline in CD11b expression (P < 0.05) and a further decrease in CD16 expression compared with preexercise values (P < 0.01). CD16 expression was lower 1 h after HI than 1 h after MI (P < 0.01). Immediately after HI, intracellular myeloperoxidase concentration was less than preexercise values (P < 0.01), whereas plasma myeloperoxidase concentration was greater (P < 0.01), indicating that HI stimulated neutrophil degranulation. Plasma myeloperoxidase concentration was higher immediately after HI than after MI (P < 0.01). Neutrophil respiratory burst activity increased after HI (P < 0.01). In summary, both MI and HI reduced neutrophil surface receptor expression. Although CD16 expression was reduced to a greater extent after HI, this reduction did not impair neutrophil degranulation and respiratory burst activity.

Children’s respiratory health and daily particulate levels in ten non-urban communities

We conducted a study to assess the association between the acute respiratory health of children and the levels of particulates in communities near and away from active opencast coal mines. The study enrolled children aged 1–11 years from the general population of five socioeconomically matched pairs of nonurban communities in northern England. Diaries of respiratory events were collected for 1405 children, and information was collected on the consultations of 2442 children with family/general practitioners over the 6-week study periods during 1996–1997, with concurrent monitoring of particulate levels. The associations found between daily PM10 levels and respiratory symptoms were frequently small and positive and sometimes varied between communities. The magnitude of these associations were in line with those from previous studies, even though daily particulate levels were low, and the children were drawn from the general population, rather than from the population with respiratory problems. The associations among asthma reliever use, consultations with general practitioners, and daily particulate levels were of a similar strength but estimated less precisely. The strength of association between all respiratory health measures and particulate levels was similar in communities near and away from opencast coal mining sites.