Biomedical subjects

A Fox

Publications and source records attributed to A Fox.

At least 37 records · Page 2Linked to original sources

PubMed2000

Excitatory effects of galanin in the spinal cord of intact, anaesthetized rats.

The effects of intrathecal galanin on the activity of single wide dynamic range (WDR) spinal dorsal horn neurones were analyzed in anaesthetized adult rats. Abeta-, Adelta- and C-fibres were activated by transcutaneous electrical stimulation which also induced wind-up and post-discharge. Galanin dose-dependently (0. 15-15 nmol/50 microl) enhanced Adelta-and C-fibre evoked responses, post-discharge and wind-up. Application of (D-Thr(6), D-Trp(8, 9), 15-ol)-galanin (1-15), a putative antagonist, did not attenuate the enhanced effects evoked by galanin. (D-Thr(6), D-Trp(8, 9), 15-ol)-galanin (1-15) applied alone (0.3-30 nmol/50 microl) to a separate population of neurones inhibited Abeta responses but enhanced post-discharge and wind-up in a similar pattern to galanin. Thus, (D-Thr(6), D-Trp(8, 9), 15-ol)-galanin (1-15) behaved as an agonist in the present experiments. In conclusion, in the spinal cord of the anaesthetized rat, galanin has a purely pro-nociceptive role, since it enhanced responses evoked by high-threshold fibre stimulation, along with post-discharge and wind-up of WDR neurones.

Anesthesia, Inhalation↗

PubMed2000

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products.

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

Mass Spectrometry↗

PubMed2000

The thioredoxin system of Helicobacter pylori.

This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.

Amino Acid Sequence↗

PubMed2000

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli.

Assessment of 16S-23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5' terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S-23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.

Bacillus↗

PubMed2000

Purkinje cell degeneration and control mice: responses of single units in the dorsal cochlear nucleus and the acoustic startle response.

The cartwheel cell is the most numerous inhibitory interneuron of the dorsal cochlear nucleus (DCN). It is expected to be an important determinant of DCN function. To assess the contribution of the cartwheel cell, we examined the discharge characteristics of DCN neurons and behavioral measures in the Purkinje cell degeneration (pcd) mice, which lack cartwheel cells, and compared them to those of the control mice. Distortion product otoacoustic emissions and auditory brainstem-evoked response thresholds were similar between the two groups. Extracellularly recorded DCN single units in ketamine/xylazine-anesthetized mice were classified according to post-stimulus time histogram (PSTH) and excitatory-inhibitory response area (EI-area) schemes. PSTHs recorded in mouse DCN included chopper, pauser/buildup, onset, inhibited and nondescript types. EI-areas recorded included Types I, II, III, I/III, IV and V. There were no significant differences in the proportions of various unit types between the pcd and control mice. The pcd units had slightly lower thresholds to characteristic frequency tones; however, they had spontaneous rates, thresholds to noise, and maximum driven rates to noise that were similar to those of the control units. Pcd mice had smaller startle amplitudes, but startle latency, prepulse inhibition/augmentation and facilitation by a background tone were comparable between the two groups. From these results, we conclude that DCN function in response to relatively simple acoustic stimuli is minimally affected by the absence of the cartwheel cells. Future studies employing more complex and/or multimodal stimuli should help assess the role of the cartwheel cells.

Acoustic Stimulation↗

PubMed2000

Cervical papanicolaou smear abnormalities and Chlamydia trachomatis in sexually active adolescent females.

STUDY OBJECTIVE: To examine the effect of cervical Chlamydia trachomatis infection on the prevalence of Papanicolaou (Pap) smear abnormalities in adolescent females. DESIGN: Retrospective study performed by examination of previously obtained cervical C. trachomatis cultures and Pap smear results. SETTING: Urban adolescent health care clinic in the Bronx, New York. PARTICIPANTS: Sexually active females, aged 13 to 23 (mean age: 17.9 years), attending the clinic for evaluation of sexually transmitted diseases. INTERVENTION: Patients who had undergone a gynecological examination with performance of cervical Pap smears and culture for C. trachomatis were enrolled in the study. MAIN OUTCOME MEASURE: Determine the prevalence of cervical C. trachomatis infection and compare cervical smear abnormalities in those with and without infection. RESULTS: Of a study population of 257 females, 24 patients (9.3%) were culture positive for C. trachomatis and 58 patients (22.6%) had significant cervical smear abnormalities, i.e., atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesion (LGSIL), or high grade squamous intraepithelial lesion (HGSIL). The 24 patients infected with C. trachomatis showed the following cervical smear abnormalities: within normal limits-37.5%, benign cellular changes-41.7%, ASCUS-12. 5%, and LGSIL-8.3%. A total of 233 patients (90.7%) were culture negative for C. trachomatis and showed the following cervical smear abnormalities: within normal limits-37.3%, benign cellular changes-39.9%, ASCUS-13.3%, LGSIL-8.6%, and HGSIL-.9%. Statistical analysis suggested no significant differences between the two groups (P >.9 by the Kruskal-Wallace test). CONCLUSIONS: The isolation of C. trachomatis from the cervix of sexually active adolescent females at a single point in time does not impact on the prevalence of significant cervical smear abnormalities.

Adolescent↗

PubMed2000

Innate immunity and graft rejection.

Although innate immunity evolved to combat pathogens, increasing awareness of a pivotal role in driving and shaping adaptive immunity has prompted this review on the role of innate immunity in graft rejection. We present evidence that grafts, especially xenografts, elicit innate responses, required for adaptive immunity. Particular attention is paid to studies by ourselves and others demonstrating the important role of innate immunity in T-cell trafficking. The mechanisms by which grafts elicit innate immunity are a fertile subject for further investigation and an important target for therapeutic intervention.

Adoptive Transfer↗

PubMed2000

Intrathecally administered endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal cord neuronal responses to nociceptive stimuli in the rat.

Inflammatory processes occurring within the central nervous system (CNS) can produce 'illness induced behaviours' which include fever, sleep and the development of allodynia and hyperalgesia. Here we demonstrate the effects of the pro-inflammatory mediators, bacterial endotoxin, and rat recombinant interleukin 1 beta (rrIL-1 beta) or tumour necrosis factor-alpha (rrTNF alpha) on the integration of somatosensory information at the single neuronal level, via recordings from wide-dynamic range neurones in the dorsal horn of the spinal cord in anaesthetized rats. Intrathecal administration of E. coli lipopolysaccharide (LPS, 10 and 100 microg, i.t.) enhanced the activity of dorsal horn neurones, including facilitation of neuronal post-discharge. Intrathecal administration of IL-1 beta (5-5000 pg) or TNF-alpha (5-5000 pg) enhanced dorsal horn neuronal responses, including the acute responses to C-fibre stimulation, wind-up and post-discharge, however, the effects of IL-1 beta were more robust than those of TNF alpha. Intrathecal administration of IL-1 beta (1-1000 pg) also leads to the development of mechanical allodynia and hyperalgesia. On the other hand intrathecal application of TNF alpha did not produce changes in sensitivity to mechanical stimuli. Changes in the activity of spinal wide-dynamic range neurones induced by local inflammation may provide a pathomechanism for the clinical pathology of central pain syndrome, which can accompany CNS disease or acute CNS injury.

Animals↗

PubMed2000

Imposed vibration influences perceived tactile smoothness.

According to the duplex theory of tactile texture perception, detection of cutaneous vibrations produced when the exploring finger moves across a surface contributes importantly to the perception of fine textures. If this is true, a vibrating surface should feel different from a stationary one. To test this prediction, experiments were conducted in which subjects examined two identical surfaces, one of which was surreptitiously made to vibrate, and judged which of the two was smoother. In experiment 1, the vibrating surface was less and less often judged smoother as the amplitude of (150 Hz) vibration increased. The effect was comparable in subjects who realized the surface was vibrating and those who did not. Experiment 2 showed that different frequencies (150-400 Hz) were equally effective in eliciting the effect when equated in sensation level (dB SL). The results suggest that vibrotaction contributes to texture perception, and that, at least within the Pacinian channel, it does so by means of an intensity code.

Adolescent↗

PubMed2000

Investigation of the concentration of bacteria and their cell envelope components in indoor air in two elementary schools.

Bacterial cell envelope components are widely distributed in airborne dust, where they act as inflammatory agents causing respiratory symptoms. Measurements of these agents and other environmental factors are assessed in two elementary schools in a southeastern city in the United States. Muramic acid (MA) was used as a marker for bacterial peptidoglycan (PG), and 3-hydroxy fatty acids (3-OH FAs) were used as markers for Gram-negative bacterial lipopolysaccharide (LPS). Culturable bacteria were collected using an Andersen sampler with three different culture media. In addition, temperature (T), relative humidity (RH), and CO2 were continuously monitored. Concentrations of airborne MA and 3-OH FAs were correlated with total suspended particulate (TSP) levels. Outdoor MA (mean = 0.78-1.15 ng/m3) and 3-OH FA levels (mean = 2.19-2.18 ng/m3) were similar at the two schools. Indoor concentrations of airborne MA and 3-OH FAs differed significantly between schools (MA: 1.44 vs. 2.84 ng/m3; 3-OH FAs: 2.96 vs. 4.57 ng/m3). Although indoor MA levels were low, they were significantly related to teachers' perception of the severity of indoor air quality (IAQ) problems in their classrooms. Concentrations of CO2 correlated significantly with all bacteria measurements. Because CO2 levels were related to the number of occupants and the ventilation rates, these findings are consistent with the hypothesis that the children and teachers are sources of bacterial contamination. Many culturable bacteria present in indoor air are opportunistic organisms that can be infectious for compromised individuals, while both culturable and nonculturable bacterial remnants act as environmental toxins for both healthy and compromised individuals. Measuring the "total bacteria load" would be most accurate in assessing the biotoxicity of indoor air. Chemical analysis of MA and 3-OH FAs, when coupled with the conventional culture method, provides complementary information for assessing biocontamination of indoor air.

Air Microbiology↗

PubMed2000

Failure To detect muramic acid in normal rat tissues but detection in cerebrospinal fluids from patients with Pneumococcal meningitis.

Muramic acid serves as a marker for the presence of bacterial cell wall debris in mammalian tissues. There have been a number of controversial and sometimes conflicting results on assessing the levels of muramic acid in health and disease. The present report is the first to use the state-of-the art technique, gas chromatography-tandem mass spectrometry, to identify and quantify the levels of muramic acid in tissues. Muramic acid was not found in normal rat brain or spleen. However, when tissues were spiked with muramic acid, it was readily identified. The detection limit was <1 ng of muramic acid/100 mg (wet weight) of tissue. The levels of muramic acid reported in diseased human spleen and spleen of arthritic rats, previously injected with bacterial cell walls, were 100- to 1,000-fold higher. In the present study, muramic acid was also readily detected in the cerebrospinal fluid of patients with pneumococcal meningitis (6.8 to 3,900 ng of muramic acid/ml of cerebrospinal fluid). In summary, there can be an enormous difference in the levels of muramic acid found in different mammalian tissues and body fluids in health and disease. This report could have great impact in future studies assessing the role of bacterial cell wall remnants in the pathogenesis of certain human inflammatory diseases.

Animals↗

PubMed1999

hFOG-2, a novel zinc finger protein, binds the co-repressor mCtBP2 and modulates GATA-mediated activation.

We have identified a novel human zinc finger protein, hFOG-2, which is related to but distinct from the murine transcription factor Friend-of-GATA-1 (mFOG-1). The hFOG-2 gene was initially detected in K562 cells using a polymerase chain reaction approach with degenerate primers corresponding to zinc finger regions of mFOG-1. A murine homologue of hFOG-2 was also identified in the mouse expressed sequence tag data banks, indicating that a family of FOG genes exists in mammals. hFOG-2 appears to be widely expressed, while mFOG-1 is expressed primarily in erythroid and megakaryocytic cells and plays a fundamental role in the development of these lineages. Sequencing of the full-length hFOG-2 cDNA indicates that the interaction domains for transcription factors GATA-1 and mCtBP2 are both conserved and we have shown that this new FOG protein also physically interacts with these factors. We have demonstrated that hFOG-2, like mFOG-1, can act in concert with GATA-1 to activate gene expression from the p45 NF-E2 promoter region, but that it can also act to repress GATA-mediated activation of additional reporter constructs. Finally, we have identified a repression domain in hFOG-2 and show that repression is dependent upon the integrity of the mCtBP2 interaction motif Pro-Ile-Asp-Leu-Ser.

Alcohol Oxidoreductases↗

PubMed1999

Electrospray quadrupole mass spectrometry analysis of model oligonucleotides and polymerase chain reaction products: determination of base substitutions, nucleotide additions/deletions, and chemical modifications.

ESI FTICR mass spectrometry is the only technique currently used for accurate molecular weight analysis of PCR products above 100 bp in size. This is important in demonstrating the potential for MS in making major contributions in the molecular biology and genomics areas. In the near future, it is more likely that less expensive, more user friendly MS techniques will be used for high-throughput analyses (including MALDI TOF and ESI quadrupole). There have been numerous reports on the use of MALDI TOF. The current report is to the first to evaluate the use of ESI-quadrupole analysis of PCR products. Synthetic oligonucleotides (30 and 89 mers) and polymerase chain reaction products of varying molecular weight (62, 88, 89, and 114 bp) were analyzed by ESI using a quadrupole MS. The mass accuracy for nucleic acids in the 30-62 bp range was shown to allow determination of nucleotide substitutions and additions/deletions. For higher molecular weight PCR products (88-114 bp), the mass accuracy of ESI-MS distinguishes single or multiple nucleotide insertions/deletions. In addition, ESI quadrupole MS allows determination of molecular weight of both strands of higher molecular weight ds PCR products and can distinguish nucleotide modifications (e.g., with biotin). In conclusion, it is demonstrated that ESI-MS occupies an intermediate position (as compared to MALDI TOF and ESI FTICR) with regard to mass accuracy and resolution in analysis of nucleic acids.

Base Sequence↗

PubMed1999

Modulation of acetylcholine release from parasympathetic nerves innervating guinea-pig and human trachea by endomorphin-1 and -2.

Endomorphin-1 and -2 (mu-opioid receptor agonists) produced a concentration-dependent and naloxone-sensitive inhibition of cholinergic contractile responses in guinea-pig trachea (at 10 microM, 46.1 +/- 8.0% and 33.8 +/- 8.6%, respectively). Endomorphin-1 and -2 also inhibited electrically-evoked acetylcholine release from cholinergic nerves innervating guinea-pig (at 0.1 microM, 41.8 +/- 10.9%; at 1 microM 60.1 +/- 6.3%, respectively) and human trachea (at 10 microM, 76.2 +/- 18.1%, and 77.7 +/- 14.3%, respectively). Naloxone prevented the inhibition by endomorphin-1 and -2 in both guinea-pig and human trachea, suggesting that these peptides can inhibit cholinergic, parasympathetic neurotransmission to the airways via the activation of classical opioid receptors.

Acetylcholine↗

PubMed1999

Immunogenicity and reactogenicity in UK infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins.

The development of effective vaccines against serogroup B meningococci is of great public health importance. We assessed a novel genetically engineered vaccine containing six meningococcal class 1 (PorA) outer membrane proteins representing 80% of prevalent strains in the UK. 103 infants were given the meningococcal vaccine at ages 2, 3 and 4 months with routine infant immunisations, with a fourth dose at 12-18 months. The vaccine was well tolerated. Three doses evoked good immune responses to two of six meningococcal strains expressing PorA proteins contained in the vaccine. Following a fourth dose, larger bactericidal responses to all six strains were observed, suggesting that the initial course had primed memory lymphocytes and revaccination stimulated a booster response. This hexavalent PorA meningococcal vaccine was safe and evoked encouraging immune responses in infants. Vaccines of this type warrant further development and evaluation.

Antibodies, Bacterial↗

PubMed1999

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry.

Bacterial cellular polysaccharides are composed of a variety of sugar monomers. These sugars serve as chemical markers to identify specific species or genera or to determine their physiological status. Some of these markers can also be used for trace detection of bacteria or their constituents in complex clinical or environmental matrices. Analyses are performed, in our hands, employing hydrolysis followed by the alditol acetate derivatization procedure. Substantial improvements have been made to sample preparation including simplification and computer-controlled automation. For characterization of whole cell bacterial hydrolysates, sugars are analyzed by gas chromatography-mass spectrometry (GC-MS). Simple chromatograms are generated using selected ion monitoring (SIM). Using total ion GC-MS, sugars can be readily identified. In more complex clinical and environmental samples, markers for bacteria are present at sufficiently low concentrations that more advanced instrumentation, gas chromatography-tandem mass spectrometry (GC-MS-MS), is preferred for optimal analysis. Using multiple reaction monitoring, MS-MS is used (replacing more conventional SIM) to ignore extraneous chromatographic peaks. Triple quadrupole and ion trap GC-MS-MS instruments have both been used successfully. Absolute chemical identification of sugar markers at trace levels is achieved, using MS-MS, by the product spectrum.

Acetates↗

PubMed1999

Sheep perforin: identification and expression by gammadelta T cells from pregnant sheep uterine epithelium.

GammadeltaTCR-positive intraepithelial lymphocytes (IEL) in sheep uterus have been shown in previous studies to express CD8 and to contain prominent intracytoplasmic granules, indicating that they may be cytotoxic. Sheep perforin, which has not previously been described, was identified in the present study using RT-PCR based on primers from human and mouse perforin sequences. A 290 base pair (bp), partial sheep perforin sequence was obtained which showed 80.3% and 66.2% nucleotide identity with human and mouse perforin, respectively. GammadeltaTCR+ IEL from sheep pregnant uteri were found to express perforin mRNA providing further evidence that these cells are cytotoxic.

Animals↗

PubMed1999

Meningococcal serogroup C conjugate vaccine is immunogenic in infancy and primes for memory.

The safety, immunogenicity, and immunologic priming of 2 dosages (2 microgram or 10 microgram) of a meningococcal C oligosaccharide-CRM197 conjugate vaccine was evaluated in 114 infants vaccinated at ages 2, 3, and 4 months. Antibody persistence and response to boosting with 10 microgram of meningococcal C polysaccharide were assessed. The meningococcal conjugate vaccine produced fewer local reactions than concurrent routine immunizations. Total serogroup C-specific immunoglobulin geometric mean concentration (GMC) increased from 0.3 microgram/mL before vaccination to 13.1 microgram/mL at age 5 months. Serum bactericidal antibody (SBA) geometric mean titers (GMTs) rose from <1:4 to 1:1057 at 5 months and fell by 14 months to 1:19. Following boosting, anti-C-specific immunoglobulin GMC rose to 15.9 microgram/mL and SBA GMT to 1:495. Antibody responses in the 10-microgram dose cohort were significantly higher at 5 months (P<.01) than in the 2-microgram dose cohort but were lower after polysaccharide boosting (P=.02). This meningococcal conjugate vaccine was well tolerated and immunogenic and induced immunologic memory in infants.

Bacterial Capsules↗