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Biomedical subjects

G J Hughes

Publications and source records attributed to G J Hughes.

At least 19 recordsLinked to original sources
PubMed2006

Experimental infection of big brown bats (Eptesicus fuscus) with Eurasian bat lyssaviruses Aravan, Khujand, and Irkut virus.

Here we describe the results of experimental infections of captive big brown bats (Eptesicus fuscus) with three newly isolated bat lyssaviruses from Eurasia (Aravan, Khujand, and Irkut viruses). Infection of E. fuscus was moderate (total, 55-75%). There was no evidence of transmission to in-contact cage mates. Incubation periods for Irkut virus infection were significantly shorter (p < 0.05) than for either Aravan or Khujand virus infections. In turn, quantification of viral RNA by TaqMan PCR suggests that the dynamics of Irkut virus infection may differ from those of Aravan/Khujand virus infection. Although infectious virus and viral RNA were detected in the brain of every rabid animal, dissemination to non-neuronal tissues was limited. Levels of viral RNA in brain of Aravan/Khujand virus-infected bats was significantly correlated with the number of other tissues positive by TaqMan PCR (p < 0.05), whereas no such relationship was observed for Irkut virus infection (where viral RNA was consistently detected in all tissues other than kidney). Infectious virus was isolated sporadically from salivary glands, and both infectious virus and viral RNA were obtained from oral swabs. The detection of viral RNA in oral swabs suggests that viral shedding in saliva occurred <5 days before the onset of clinical disease.

Animals↗
PubMed2006

Power Doppler ultrasound assessment of follicular vascularity in the early follicular phase and its relationship with outcome of in vitro fertilization.

OBJECTIVE: To determine whether ovarian perifollicular blood flow (PFBF) in the early follicular phase (EFP) was associated with treatment outcome. DESIGN: Retrospective longitudinal cohort study. SETTING: Tertiary referral centre/university hospital. PATIENTS: Thirty-four women underwent 37 IVF cycles, which resulted in 35 embryo transfers. INTERVENTIONS: Serial transvaginal scans using power Doppler ultrasound during the follicular phase. Ovarian PFBF of follicles > or =5 mm was subjectively assessed using a modified grading system (grades 0-4). MAIN OUTCOME MEASURES: Ovarian PFBF and pregnancy. RESULTS: Treatment cycles were retrospectively divided into two groups: Group 1 (n=20) had cycles with at least one small (5-10 mm) or medium (11-14 mm) size follicle(s) of high grade (2-4) PFBF on cycle day 5 or 6 or 7; and Group 2 (n=17), had cycles that did not. Group 1 had a significantly higher proportion of high grade large follicles in the late follicular phase (35% vs. 21%) (OR 2.0; 95% CI 1.1-3.7) and higher clinical pregnancy rate (47% vs. 12%) (OR 6.3; CI 1.1-35.7) compared to Group 2. CONCLUSION: High grade ovarian PFBF in the EFP during IVF is associated with both high grade PFBF in the late follicular phase and a higher clinical pregnancy rate.

Adult↗
PubMed2006

Phylogenetic relationships of seven previously unclassified viruses within the family Rhabdoviridae using partial nucleoprotein gene sequences.

Partial nucleoprotein (N) gene sequences of the rhabdoviruses Obodhiang (OBOV), Kotonkon (KOTV), Rochambeau (RBUV), Kern canyon (KCV), Mount Elgon bat (MEBV), Kolongo (KOLV) and Sandjimba (SJAV) were generated and their phylogenetic positions within the family Rhabdoviridae were determined. Both OBOV and KOTV were placed within the genus Ephemerovirus. RBUV was joined to the same cluster, but more distantly. MEBV and KCV were grouped into a monophyletic cluster (putative genus) with Oita virus (OITAV). These three viruses, originating from different regions of the world, were all isolated from insectivorous bats and may be specific for these mammals. African avian viruses KOLV and SJAV were joined to each other and formed another clade at the genus level. Further, they were grouped with the recently characterized rhabdovirus Tupaia virus (TRV). Although the genetic distance was great, the grouping was supported by consistent bootstrap values. This observation suggests that viruses of this group may be distributed widely in the Old World. Non-synonymous/synonymous substitution ratio estimations (dN/dS) using a partial N gene fragment (241 codons) for the three rhabdovirus genera revealed contrasting patterns of evolution, where dN/dS values follow the pattern Ephemerovirus > Vesiculovirus > Lyssavirus. The magnitude of this ratio corresponds well with the number of negatively selected codons. The accumulation of dS appears evenly distributed along the gene fragment for all three genera. These estimations demonstrated clearly that lyssaviruses are subjected to the strongest constraints against amino acid substitutions, probably related to their particular niche and unique pathobiology.

Amino Acid Sequence↗
PubMed2004

Longitudinal assessment of ovarian perifollicular and endometrial vascularity by power Doppler ultrasound in pregnant and non-pregnant cycles in the IVF setting.

PURPOSE: This longitudinal study aimed to compare ovarian perifollicular and endometrial blood flow (PFBF and EBF, respectively) during the follicular phase in pregnant and non-pregnant IVF cycles. METHODS: Serial transvaginal scans were performed in 15 subjects undergoing IVF treatment. Both PFBF and EBF were subjectively graded (grades 0-4 for PFBF and grades 1-3 for EBF). After confirmation of clinical pregnancy, the treatment cycles were grouped into 'Pregnant' and 'Non-pregnant' cycles. Ovarian PFBF and EBF were retrospectively compared between the two groups. RESULTS: In pregnant cycles, the proportion of large (> or = 15 mm) follicles with high (24) grade PFBF increased with time throughout the follicular phase, and the proportion of large follicles with poor (0-1) grade PFBF decreased. In non-pregnant cycles these trends were reversed. There was no difference in EBF between the two groups. CONCLUSION: The pattern of ovarian PFBF but not EBF may be predictive of treatment outcome.

Adult↗
PubMed2004

Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads.

Published assays that use TaqMan PCR are consistently sensitive, rapid, and readily transferable. Here we describe a TaqMan PCR-based method for the detection of rabies virus (RV) RNA in tissue samples. We show that the method has an acceptable linear range, is both sensitive and specific, and, importantly, correlates with the concentration of infectious virus. In addition, the levels of RV-specific amplification are adjustable according to the levels of an endogenous control (beta-actin mRNA), allowing the calculation of comparable quantities. We tested the capacity of this assay to cope with target sequence variations. The number of sequence mismatches between gene-specific oligonucleotides and the target sequence significantly affects amplification (P < 0.001), and point mutations at the center of the probe can result in false-negative results through the prevention of probe binding and subsequent fluorescence. This study demonstrates that the genetic heterogeneity of RVs may prove a serious obstacle in the development of a diagnostic assay based on TaqMan PCR; however, the quantification of RV levels may prove to be a valuable application of this assay.

Animals↗
PubMed2002

Cerebrospinal fluid concentrations of atracurium, laudanosine and vecuronium following clinical subarachnoid hemorrhage.

BACKGROUND: Neuromuscular blocking agents may exert central nervous system effects when they reach the brain. This study assessed the concentrations and the time course of passage of vecuronium, atracurium, and its metabolite laudanosine in the cerebrospinal fluid (CSF) of patients undergoing intracranial aneurysm clipping. METHODS: Twenty-five patients with subarachnoid hemorrhage were randomly allocated to receive an intravenous infusion of vecuronium (n=13) or atracurium (n=12). Arterial blood and lumbar CSF were sampled before and 1, 2, 3, 4 and 8 h after the start of the relaxant infusion. The samples were analyzed by liquid chromatography-electrospray ionization mass spectrometry (vecuronium) and high-pressure liquid chromatography (atracurium and laudanosine). RESULTS: The data of 20 patients (10 in both groups) were analyzed. In 11 CSF samples from five patients atracurium was detected in concentrations from 10 to 50 ng/ml. Laudanosine was retrieved in all CSF samples at 1, 2, 3, 4 and 8 h; the highest CSF concentration of laudanosine occurred at 3 h [38 (18-63) ng/ml: median (range)]. Vecuronium was not found in any CSF sample. CONCLUSION: Significant concentrations of atracurium and laudanosine but not of vecuronium were detected in the CSF of patients during and immediately after intracranial aneurysm surgery.

Adolescent↗
PubMed2002

Dose-dependent responses of sheep inoculated intranasally with a type O foot-and-mouth disease virus.

Unlike foot-and-mouth disease (FMD) in cattle and pigs, which spreads rapidly, resulting in easily detectable foci of clinical infection, the disease in sheep is characterized by restricted transmission, low morbidity and sporadic clinical cases. The study described was designed to investigate whether the ability of sheep to transmit and maintain FMD virus was dose-related. The viral isolate used was known to be associated epidemiologically with rapid fade-out of transmission within sheep flocks. Five separate transmission experiments were performed, with different doses of FMD virus, each experiment containing five intranasally inoculated donor sheep and 10 in-contact recipient sheep. The lowest dose required to cause clinical infection by inoculation (10(4) 50% tissue culture infectious doses; 10(4) TCID50) was also the optimum dose for producing in-contact transmission. Inoculation of donor sheep with higher doses (10(5) and 10(6) TCID50) resulted in reduced transmission, characterized by reduced duration and degree of viraemia and an early humoral and cell-mediated immune response. Principal component analysis was used to interpret the complex interactions of the dose-related responses to infection.

Administration, Intranasal↗
PubMed2002

Clinical variation in foot and mouth disease: sheep and goats.

Foot and mouth disease (FMD) in adult sheep and goats is frequently mild or unapparent, but can cause high mortality in young animals. The recent outbreak of FMD in the United Kingdom has highlighted the importance of sheep in the epidemiology of the disease, although there have been numerous examples in the past where small ruminants have been responsible for the introduction of FMD into previously disease-free countries. The difficulty in making a clinical diagnosis should encourage the development of more rapid screening tests to assist in future control programmes.

Animals↗
PubMed2001

Api m 6: a new bee venom allergen.

BACKGROUND: Characterization of the primary structure of allergens is a prerequisite for the design of new diagnostic and therapeutic tools for allergic diseases. OBJECTIVE: The purpose of this study was the identification and characterization of a low-molecular-weight, IgE-binding, bee venom (BV) allergen. METHODS: BV proteins were separated by using size exclusion chromatography and HPLC. IgE antibody binding to purified proteins was analyzed by means of immunoblotting, and T-cell response was analyzed by means of proliferation assay. Amino acid sequence was determined with 2 approaches, namely Edman degradation and carboxy terminal analysis with mass spectrometry. RESULTS: Api m 6, which migrated as an 8-kd band in SDS-PAGE, was frequently (42%) recognized by IgE from BV-hypersensitive patients. In addition, PBMCs from BV-hypersensitive patients, as well as from a normal control subject, proliferated in response to this allergen. Api m 6 exists as 4 isoforms of 7190, 7400, 7598, and 7808 d, respectively. Amino acid sequences obtained from HPLC-purified preparations revealed that the isoforms were constituted of a common central core of 67 residues, only differing in the amino- and carboxy-terminal ends. Api m 6 showed no significant sequence homology with known proteins. CONCLUSIONS: We have identified and sequenced a new BV allergen that elicits a strong IgE and T-cell response in a large number of BV-hypersensitive patients. Api m 6 should be considered in the diagnostic and therapeutic approach of BV immunotherapy on the basis of peptides or recombinant proteins.

Allergens↗
PubMed2001

Prognostic value of baseline serum oestradiol in controlled ovarian hyperstimulation of women with unexplained infertility.

This study aimed to evaluate the prognostic value of the baseline serum oestradiol E2 level on the cycle pregnancy rate (PR) in women with unexplained infertility (UI) undergoing controlled ovarian hyperstimulation (COH). This study is unique in that it evaluates the effect of cycle day 1 (rather than day 3) E2 levels on cycle PR in a COH setting (without IVF) in women with UI (rather than multiple causes of infertility). Structured as a retrospective cohort study, the setting was the Ovulation Induction Clinic at an academic tertiary care hospital. One hundred and forty-five patients with UI underwent 374 cycles of COH with either human menopausal gonadotrophin (hMG) alone or hMG and clomiphene citrate. Outcome was measured as cycle pregnancy rate (PR) according to the cycle day 1 level of E2. Patients with an E2 level > 150 pmol/l on cycle day 1 of COH achieved a significantly lower PR (4%) compared with those with E2 levels < or = 150 pmol/l (13%). Logistic regression analysis demonstrated that women with day 1 E2 levels below 150 pmol/l were 3.2 times more likely to conceive than those with day 1 E2 levels above 150 pmol/l. Also, the impact of day 1 E2 levels on the chance of pregnancy was independent of day 1 serum FSH levels. Women with UI undergoing COH in our unit with an elevated baseline serum E2 > 150 pmol/l have a significantly lower PR and should be counselled regarding the decreased likelihood of pregnancy.

Adult↗
PubMed2000

Formation of peptide impurities in polyester matrices during implant manufacturing.

Most peptides are susceptible, in vivo, to proteolytic degradation, and it is difficult to formulate and to deliver them without loss of biological activity. In addition, it is often desirable to release them continuously and at a controlled rate over a period of weeks or months. For these reasons, a controlled release system is suitable. Poly(lactic acid) (PLA) is a biocompatible and biodegradable material that can be used for many applications, including the design of injectable controlled release systems for pharmaceutical agents. Development of these delivery systems presents challenges in the assessment of stability, specially for peptide drugs. By means of an extrusion method, long-acting poly(lactic acid) implants containing vapreotide, a somatostatin analogue, were prepared. The nature of the main degradation product obtained after implant manufacturing was elucidated. It was found that the main peptide impurity was a lactoyl lactyl-vapreotide conjugate. Because lactide are found in small quantities in most commercially available PLA, the influence of residual lactide in the polymeric matrix, on the formation of peptide impurities during manufacturing, was specially investigated. This work demonstrates that the degree of purity of the carrier is of great importance with regard to the formation of peptide impurities.

Analgesics↗
PubMed1999

Carbohydrate moieties in human secretory component.

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.

Amino Acid Sequence↗
PubMed1999

Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.

A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.

Amino Acid Sequence↗
PubMed1999

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.

Carcinoma, Renal Cell↗
PubMed1999

IgE and T-cell responses to high-molecular weight allergens from bee venom.

BACKGROUND: Bee venom contains multiple allergens with a wide distribution of molecular weight. In contrast with conventional bee venom desensitization, peptide or recombinant allergen immunotherapy may have to take into account patients' individual patterns of humoral or cellular response. OBJECTIVE: To study immunoglobulin (Ig)E and T-cell responses to high-molecular weight bee venom allergens >/= 50 kDa. METHODS: Bee venom proteins were separated by size exclusion chromatography and fractions were characterized by one and two-dimensional gel electrophoresis. IgE antibody binding to bee venom fractions was analysed by immunoblotting and T-cell responses by proliferation assay. RESULTS: Among 38 bee venom-hypersensitive patients, IgE recognition pattern of bee venom allergens varied greatly. IgE bound mainly to phospholipase A2 and furthermore to several proteins >/= 50 kDa (50, 54, 69, 84 and 94 kDa). N-terminal sequences of these proteins showed no homology with known proteins. In addition, peripheral mononuclear cells from patients as well as from nonatopic donors strongly proliferated in response to those proteins. CONCLUSIONS: Although present in low amounts, high-molecular weight allergens from bee venom elicit strong IgE and T-cell responses, and may need to be considered as clinically relevant. Therefore, the development of peptide or recombinant protein-based immunotherapy for bee venom allergy may require careful characterization of such allergens.

Allergens↗
PubMed1998

Thiol-disulfide redox buffers maintain a structure of immunoglobulin A that is essential for optimal in vitro binding to secretory component.

We have shown that human secretory component (SC) binds in vitro to different samples of human and murine dimeric immunoglobulin A (IgA). The binding ratio in the IgA/SC complex is 1:1. IgA which is stably bound to SC is separated from unreacted IgA by anion exchange chromatography. A part of IgA/SC complexes formed in vitro is unstable to this elution; the proportion varies between different samples of IgA; it increases following prolonged incubation of IgA at 37 degrees C. Incubation of IgA with glutathione/glutathione disulfide (GSH/GSSG) redox buffers increases the proportion able to form a stable complex with SC to approximately 90%. The presence of bound SC is not essential for this process but does allow it to occur at a lower GSH/GSSG concentration. The stable IgA/SC complex consists of a structure with a disulfide bond between IgA and SC apparently in equilibrium with a structure in which this bond is absent. The proportion bound covalently is similar for different samples of IgA and is insensitive to incubation with GSH/GSSG. It is significantly greater for secretory IgA (sIgA) and for IgA and SC incubated together with a starting mixture of cysteine/cystine. Monoclonal, antigen-specific IgA, all of which is optimally bound to SC in essentially the same way as in native sIgA, can be isolated in high yield. Our results support a mechanism for optimal binding of IgA to SC, that can occur both in vitro and in vivo, in which a thiol disulfide interchange occurs between a free IgA thiol and a sensitive SC disulfide following the initial non-covalent interaction.

Anion Exchange Resins↗
PubMed1998

'98 Escherichia coli SWISS-2DPAGE database update.

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Bacterial Proteins↗