bluuebunny/crossref_metadata_2025 huggingface dataset:
Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic Bacillus sp.
Authors: Nath Devyani , Rao Mala
Published: March 2003

To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 m guanidine hydrochloride with an emission maximum of 345 nm; the far‐UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1‐anilinonapthalene‐8‐sulfonate (1,8‐ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two‐step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl‐β‐cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The ‘delayed detergent addition’ experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.
Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes
Authors: Pascussi Jean‐Marc , Drocourt Lionel , Gerbal‐Chaloin Sabine , Fabre Jean‐Michel , Maurel Patrick , Vilarem Marie‐José
Published: March 2003

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm–100 µm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low‐dexamethasone component (nmol concentrations) of low amplitude (factor of 3–4) followed by a high‐dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15–30). We show that the low‐dexamethasone component results from the glucocorticoid receptor‐mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic‐independent manner. At supramicromolar concentrations (>10 µm), dexamethasone binds to and activates pregnane X receptor thus producing the high‐dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).
Production and chemiluminescent free radical reactions of glyoxal in lipid peroxidation of linoleic acid by the ligninolytic enzyme, manganese peroxidase
Authors: Watanabe Takashi , Shirai Nobuaki , Okada Hitomi , Honda Yoichi , Kuwahara Masaaki
Published: March 2003

Glyoxal is a key compound involved in glyoxal oxidase (GLOX)‐dependent production of glyoxylate, oxalate and H2O2 by lignin‐degrading basidiomycetes. In this paper, we report that glyoxal was produced from a metabolite of ligninolytic fungi, linoleic acid, by manganese peroxidase (MnP)‐dependent lipid peroxidation. In the absence of the parent substrate of linoleic acid, the dialdehyde was oxidized by MnP and Mn(III) chelate to start free radical reactions with emission of chemiluminescence at 700–710 nm. The spectroscopic profile of the light emission is distinguishable from (a) singlet oxygen, (b) triplet carbonyls from dioxetane and α‐hydroxyperoxyl radicals, and (c) biacyl triplet formed by the coupling of two acyl radicals. The photon emission of glyoxal by MnP was activated by co‐oxidation of tartrate. The MnP‐dependent oxidation of glyoxal in tartrate buffers continued for 10 days without addition of exogenous H2O2. The importance of these results is discussed in relation to the free radical chemistry of lignin biodegradation by wood rot fungi.
The regulatory subunit of a cGMP‐regulated protein kinase A of Trypanosoma brucei
Authors: Shalaby Tarek , Liniger Matthias , Seebeck Thomas
Published: March 2003

This study reports the identification and characterization of the regulatory subunit, TbRSU, of protein kinase A of the parasitic protozoon Trypanosoma brucei. TbRSU is coded for by a single copy gene. The protein contains an unusually long N‐terminal domain, the pseudosubstrate site involved in binding and inactivation of the catalytic subunit, and two C‐terminally located, closely spaced cyclic nucleotide binding domains. Immunoprecipitation of TbRSU coprecipitates a protein kinase activity with the characteristics of protein kinase A: it phosphorylates a protein kinase specific substrate, and it is strongly inhibited by a synthetic protein kinase inhibitor peptide. Unexpectedly, this kinase activity could not be stimulated by cAMP, but by cGMP only. Binding studies with recombinant cyclic nucleotide binding domains of TbRSU confirmed that both domains bind cGMP with Kd values in the lower micromolar range, and that up to a 100‐fold excess of cAMP does not compete with cGMP binding.
Structural and biochemical characterization of neuronal calretinin domain I–II (residues 1–100)
Authors: Palczewska Małgorzata , Groves Patrick , Ambrus Attila , Kaleta Agata , Kövér Katalin E. , Batta Gyula , Kuźnicki Jacek
Published: March 2003

This study characterizes the calcium‐bound CR I–II domain (residues 1–100) of rat calretinin (CR). CR, with six EF‐hand motifs, is believed to function as a neuronal intracellular calcium‐buffer and/or calcium‐sensor. The secondary structure of CR I–II, defined by standard NMR methods on13C,15N‐labeled protein, contains four helices and two short interacting segments of extended structure between the calcium‐binding loops. The linker between the two helix–loop–helix, EF‐hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I–II) confirms that the linker of CR I–II is solvent‐exposed and that other potential sites are protected by regular secondary structure.45Ca‐overlay of glutathione S‐transferase (GST)–CR(1–60) and GST–CR(61–100) fusion proteins confirm that both EF‐hands of CR I–II have intrinsic calcium‐binding properties. The primary sequence and NMR chemical shifts, including calcium‐sensitive glycine residues, also suggest that both EF‐hand loops of CR I–II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross‐linking and NMR translation diffusion measurements indicate that CR I–II exists as a monomer. Calb I–II (the homologous domain of calbindin D28k) has the same EF‐hand secondary structures as CR I–II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999)Eur. J. Biochem.262, 933–938]. In contrast, Calb I–II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I–II and Calb I–II probably have distinct tertiary structure features that suggest different cellular functions for the full‐length proteins.
Structural determinants of the half‐life and cleavage site preference in the autolytic inactivation of chymotrypsin
Authors: Bódi Árpád , Kaslik Gyula , Venekei István , Gráf László
Published: March 2003

The molecular mechanism of the autolysis of rat α‐chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site‐directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114→Ile and Tyr146→His/Asn147→Ser, that had the same enzymatic activity and molecular stability as the wild‐type enzyme, displayed altered routes of autolytic degradation. The Phe114→Ile mutant also exhibited a significantly slower autolytic inactivation (its half‐life was 27‐fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild‐type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146‐Asn147 → Phe114 → other sites; (b) the cleavage rates at sites Phe114 and Tyr146‐Asn147 were independent from each other; and (c) the hydrolysis of the Phe114‐Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half‐life of chymotrypsin activity.
An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo‐electron microscopy studies
Authors: Ford Robert C. , Stoylova Svetla S. , Holzenburg Andreas
Published: March 2003

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked ‘grana’ regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo‐electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.
Induction of chicken ovalbumin upstream promoter‐transcription factor I (COUP‐TFI ) gene expression is mediated by ETS factor binding sites
Authors: Salas Ramiro , Petit Fabrice G. , Pipaon Carlos , Tsai Ming‐Jer , Tsai Sophia Y.
Published: March 2003

Chicken ovalbumin upstream promoter‐transcription factor␣I (COUP‐TFI, or NR2F1) is an orphan nuclear receptor that plays a major role in the development of the nervous system. We show here that three ETS response elements in the COUP‐TFI promoter mediate its transcription. A reporter gene containing these ETS binding sites is activated by Ets‐1, while the same reporter with point mutations on all three ETS response elements is not. We also show that Ets‐1 binds to these response elements and that other ETS factors also transactivate the COUP‐TFI promoter. In addition, COUP‐TFI is coexpressed with some ETS factors in the mouse embryo. These results indicate that members of the ETS family can activate COUP‐TFI gene expression.
Amphibian peptides that inhibit neuronal nitric oxide synthase
Authors: Doyle Jason , Llewellyn Lyndon E. , Brinkworth Craig S. , Bowie John H. , Wegener Kate L. , Rozek Tomas , Wabnitz Paul A. , Wallace John C. , Tyler Michael J.
Published: March 2003

Two neuropeptides have been isolated and identified from the secretions of the skin glands of the Stony Creek Frog Litoria lesueuri. The first of these, the known neuropeptide caerulein 1.1, is a common constituent of anuran skin secretions, and has the sequence pEQY(SO3)TGWMDF‐NH2. This neuropeptide is smooth muscle active, an analgaesic more potent than morphine and is also thought to be a␣hormone. The second neuropeptide, a new peptide, has been named lesueurin and has the primary structure GLLDILKKVGKVA‐NH2. Lesueurin shows no significant antibiotic or anticancer activity, but inhibits the formation of the ubiquitious chemical messenger nitric oxide from neuronal nitric oxide synthase (nNOS) at IC50 (16.2 µm), and is the first amphibian peptide reported to show inhibition of nNOS. As a consequence of this activity, we have tested other peptides previously isolated from Australian amphibians for nNOS inhibition. There are three groups of peptides that inhibit nNOS (IC50 at µm concentrations): these are (a) the citropin/aurein type peptides (of which lesueurin is a member), e.g. citropin 1.1 (GLFDVIKKVASVIGGL‐NH2) (8.2 µm); (b) the frenatin type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLGGLFKPK‐OH) (6.8 µm); and (c) the caerin 1 peptides, e.g. caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL‐NH2) (1.7 µm). From Lineweaver–Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine. When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca2+calmodulin, the inhibition dropped by ≈ 50% in each case. In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca2+ calmodulin. It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca2+calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS.
The sizes, maturity stages and biomass of mayfly assemblages colonizing disturbed stream bed patches in central Kenya
Authors: Mathooko Jude Mutuku
Published: March 2003

The sizes, maturity stages and biomass of mayfly species colonizing disturbed patches on the stream bed surface of the Naro Moru River, Kenya, were determined from June 1993 to January 1994. All maturity stages I–VI+ were present in the pre‐disturbance mayfly assemblages. Colonization of the disturbed patches by mayflies of different maturity stages was continuous, but slow or fast depending on the season and species. The overall post‐disturbance maturity structure of the mayflies colonizing the disturbed patches did not demonstrate any distinct pattern. The majority of Caenis nymphs colonizing the patches in wet season II (November–January) were in emerging maturity stage VI+, whilst the majority of the individuals of Afronurus and Choroterpes (Euthraulus) populations matured from stage I in the wet seasons to stage VI+ in the dry season. Furthermore, the majority of the individuals of Baetis (Nigrobaetis) sp.1 were maturing to stages IV–VI during the wet seasons. Small‐sized mayfly individuals (body length <3.0 mm) of all mayflies colonized the disturbed patches in the majority of numbers. However, although having the highest densities, they contributed a very low biomass compared with the fewer large‐sized nymphs. There was no size gradation in the colonization of the disturbed patches, since all sizes were sampled at all times but in differing proportions. Colonization of the patches by mayflies was not size‐ or maturity stage‐specific. Size distribution patterns could be useful in assessing which mayfly size‐spectra are more or less susceptible to disturbance in streams.
The demise of the Aral Sea – an environmental disaster
Authors: Waltham Tony , Sholji Ihsan
Published: March 2003

The shrinkage of the Aral Sea ranks as one of the world's greatest man‐made environmental disasters. While there is some hope for a separated northern part, the main sea is doomed
Host plant and ants influence the honeydew sugar composition of aphids
Authors: Fischer M. K. , Shingleton A. W.
Published: March 2003

Honeydew composition is an important factor in mediating ant–homopteran mutualisms. The trisaccharide melezitose is especially significant in this interaction. Honeydew of threeChaitophorusaphids (Homoptera: Aphididae), reared on two species ofPopulus(Salicaceae), was analysed to investigate factors that may influence honeydew composition.

 Honeydew ofChaitophorus populialbaeandC. populeticontained high proportions of the melezitose. These species are typically tended by ants. In contrast,C. tremulaehoneydew contained low proportions of melezitose. This species is typically untended.

 Chaitophorus populetiandC. populialbaeproduced higher proportions of melezitose when reared onPopulus tremulathan onP. alba.

 Chaitophorus populetiandC. populialbaereduced their production of melezitose when reared in the absence of ants. The reverse was true inC. tremulae.

 These results show that honeydew composition differs not only between aphid species but also within aphid species. They support known differences in ant preference for the different aphid species, and produce further predictions for ant preference for aphids on different host plants. In particular they show for the first time that ant tending may itself influence honeydew composition. They suggest that maintaining a honeydew composition attractive to ants may incur metabolic costs on aphids.
Natural variability in zygote dispersal ofAscophyllum nodosumat small spatial scales
Authors: Dudgeon S. , Kübler J. E. , Wright W. A. , Vadas Sr R. L. , Petraitis P. S.
Published: March 2003

Dispersal of propagules of marine benthic species may contribute to variation in the structure and dynamics of populations.Ascophyllum nodosumdominates the mid‐intertidal zone of sheltered rocky shores in the north‐west Atlantic, and populations often consist of large, mature individuals and few newly recruited thalli.

 We investigated the dispersal of zygotes ofA. nodosumat three sites on Swans Island, ME, USA, to determine its variability under natural conditions and to test whether limited dispersal explains recruitment patterns.

 At each site we released artificially fertilized, marked zygotes from a single point during the rising tide, and sampled their dispersal using two methods: sampling the water column to track the dispersing cloud of zygotes; and assaying caged ceramic tiles affixed to the shore for the presence of marked zygotes.

 At all sites, zygotes were diluted to ≤1·3% of the initial concentration within 8–12 min of their release. The mean direction of transport was correlated with both wind and tidal current direction at only one site. The most dense settlement occurred at this site, suggesting greater local reproductive success.

 Regression analyses based on settlement densities suggest that a quadratic model fit the data best for all sites. Quadratic terms occurred in the direction normal to flow, and are interpreted to represent turbulent transport and diffusion. Both linear and quadratic terms were significant parallel to flow, and we ascribe these to advective transport without and with turbulent mixing, respectively.

 The range of zygote dispersal exceeded 6 m from the source, and therefore cannot explain the exponential decline of recruits in large patches (≈10 m diameter) within adult stands ofA. nodosum.

 Migration of zygotes between populations is unlikely, but their local dispersal may strongly influence the spatial and temporal dynamics within populations.
Effects of maternal identity and incubation temperature on Snapping Turtle (Chelydra serpentina) growth
Authors: Steyermark A. C. , Spotila J. R.
Published: March 2003

Individual variation in physiological traits may have important consequences for offspring survival rates and adult fitness. Variance in offspring phenotypes is due to interindividual differences in genotype, environment or maternal effects. This study examined the contributions of family identity, egg incubation environment and maternal effects to individual variation in growth rates in the common Snapping Turtle,Chelydra serpentina.

 The growth rates of 254 individuals, representing 24 clutches, were measured for approximately 6 months.

 Mean growth rate for all individuals was 7·21 mg g−1 day−1(= day−1) (SE = 0·12 day−1, range =−3·70–13·03 day−1). Growth rates varied significantly among clutches, ranging from 5·76 to 9·20 day−1, indicating that family identity, and possibly genotype, significantly affects posthatching growth. In general, larger eggs produced larger hatchlings, and individuals that were larger at birth were larger at 175 days of age. Thus, maternal effects were significant throughout the entire growth measurement period.

 In contrast to results from previous studies, egg incubation temperature did not significantly affect individual growth.

 Because all individuals received similar treatments and were in similar environments posthatching, our results suggest that there is a genetic component to growth rate, but that egg incubation temperature does not affect growth.
Predicting daytime carbon isotope ratios of atmospheric CO2 within forest canopies
Authors: Buchmann N. , Brooks J. R. , Ehleringer J. R.
Published: March 2003

While measurements of leaf carbon isotope ratios (13C/12C) in terrestrial ecosystems have become more frequent, interpreting these data can remain a challenge in well developed canopies: the variation in leaf δ13C (δ13Cp) values is influenced by both the variation in δ13C of source air (δ13Ca) and by photosynthetic carbon isotope discrimination (Δ). However, source air information is often unavailable, limiting the interpretation of δ13Cp, particularly in dense stands.

  In this synthesis we found that about 70% of the observed variation in δ13Cp values within the canopy was influenced by changes in Δ, and that about 30% was determined by source air effects. Significant shifts in δ13Ca occur in canopies with high leaf area, predominantly within 1 m above the forest floor. In complex canopies, particularly in the understorey, source air effects cannot be neglected if δ13Cp measurements are used to calculate Δ and ci/ca ratios [ratio of internal CO2 concentration in the mesophyll airspaces (ci) to the ambient atmospheric concentration of CO2 (ca)].

  We modelled δ13Ca of daytime source air for deciduous and coniferous forests in boreal, temperate and tropical biomes. An inverse regression model with easily available input variables accounted for about 90% of the variation in daytime δ13Ca values throughout the canopy.

  In open canopies with leaf area index (L) of <2·5 or at canopy heights ≥1 m, the within‐canopy daytime δ13Ca differences are negligible, and variations in δ13Cp are associated primarily with changes in Δ. Then, one can use the easily available carbon isotope ratio of the troposphere (δ13Ctrop) as a substitute for δ13Ca to calculate Δ to within ±0·4‰.

  In canopies with L values >2·5, and at canopy heights <1 m, our model is recommended for calculating canopy δ13Ca values when direct measurements are not feasible. Although δ13Ca is highly variable near the forest floor in those dense forests, our model is more accurate and precise for estimating δ13Ca within 1 m above the forest floor than using δ13Ctrop throughout the canopy (−0·2‰ ± 1·5 versus −1·4‰ ± 1·1).
Metabolic recovery in the Desert Iguana (Dipsosaurus dorsalis) following activities of varied intensity and duration
Authors: Hancock T. V. , Gleeson T. T.
Published: March 2003

Elevated oxygen consumption during recovery is the majority of total metabolic costs associated with short vigorous activity, thus any assessment of total costs associated with such activity should also examine the excess postexercise oxygen consumption (EPOC). This study examined EPOC and total metabolic costs in Dipsosaurus (N = 8; 75 ± 3 g) over a variety of submaximal intensities: 50%, 100% and 400% of the maximum aerobic speed (MAS = 0·27 m s−1; the speed at which V̇O2max is elicited). Activity duration was also varied (15, 60 and 300 s) over these intensities.

  Total costs increased significantly with both activity intensity and duration. EPOC increased significantly with activity intensity. EPOC increased significantly with activity duration at higher intensities, but not the lowest intensity of 50% MAS.

  These recovery costs constitute amounts of energy large enough to have an impact on energy budget analyses. The metabolic cost of an individual bout of activity and subsequent recovery was as high as 5% of the predicted daily energy expenditure, an increase of 5–8 × estimates that do not incorporate recovery.

  To compare the relative economy of different activity regimens, the cost of activity (Cact; ml O2 g−1 km−1), was calculated as total costs, including EPOC, per unit distance travelled. Cact was independent of activity intensity over these ranges, but was inversely related to activity duration.

  From these data, the most economical strategy to travel a certain distance would utilize a low‐intensity and extended duration, as seen in regular activities such as foraging. High‐intensity activities, such as escape, prey capture or mating behaviours, are thus predicted to be less economical. These should only be observed where metabolic costs would be considered secondary to the more proximal needs of the animal.
The effects of steady‐state erythromycin and azithromycin on the pharmacokinetics of sildenafil in healthy volunteers
Authors: Muirhead Gary J. , Faulkner Stephen , Harness Jane A. , Taubel Jörg
Published: March 2003

Aims  Sildenafil, an effective oral treatment for erectile dysfunction, is predominantly metabolized by the cytochrome P450 isozyme 3A4, which is inhibited by a number of the macrolide antibiotics. Therefore, two placebo‐controlled, parallel‐group studies were conducted to evaluate the effects of multiple doses of erythromycin and azithromycin on the pharmacokinetics, safety and tolerability of a single oral 100‐mg dose of sildenafil.

Methods  In the erythromycin interaction study, 26 male volunteers (18–45 years of age) received open‐label sildenafil 100 mg on day 1. Half received blinded erythromycin (500 mg) twice daily on days 2–6, and the other half received placebo. On day 6, all subjects received a second 100‐mg dose of sildenafil. In the azithromycin interaction study, 24 male volunteers (19–33 years of age) received open‐label 100 mg sildenafil on day 1. Half then received blinded azithromycin (500 mg) once daily on days 2–4, and the other half received placebo. On day 4, all subjects received another 100‐mg dose of sildenafil. In both studies, blood samples were collected on the first and last study day for the analysis of plasma concentrations of sildenafil and its primary metabolite, UK‐103,320.

Results  Repeated dosing with erythromycin caused statistically significant increases in the AUC and Cmax of sildenafil (2.8‐fold and 2.6‐fold, respectively) but had no effect on Tmax, kel or t1/2. A statistically significant 1.4‐fold increase in the AUC of UK‐103,320 was also observed, as well as a significant decrease in kel, resulting in an increase of about 1 h in t1/2. In contrast, repeated dosing with azithromycin caused no significant change in any pharmacokinetic parameter of either sildenafil or UK‐103,320. Erythromycin, azithromycin and sildenafil were well tolerated; adverse events were mild and transient. No subject withdrew from either trial for any reason related to study drug.

Conclusions  These results indicate that erythromycin modifies the pharmacokinetics of sildenafil by inhibiting its CYP3A4‐mediated first‐pass metabolism. Given these data, a lower starting dose of sildenafil (25 mg) may be considered for patients receiving erythromycin or other potent CYP3A4 inhibitors. Azithromycin did not affect the pharmacokinetics of sildenafil; therefore, no adjustment in dosage is necessary for patients receiving these drugs concomitantly.
Upper gastrointestinal bleeding among users of NSAIDs: a population‐based cohort study in Denmark
Authors: Mellemkjær Lene , Blot William J. , Sørensen Henrik Toft , Thomassen Lars , McLaughlin Joseph K. , Nielsen Gunnar Lauge , Olsen Jørgen H.
Published: March 2003

Aims  It is well‐known that use of nonsteroidal anti‐inflammatory drugs (NSAIDs) increases the risk of upper gastrointestinal bleeding (UGIB), but characteristics of the association and quantification of excess risk at the population level require clarification.

Methods  All users of nonaspirin prescription NSAIDs in North Jutland County, Denmark during 1991–95 were identified in the regional Pharmaco‐Epidemiologic Database. Using the Hospital Discharge Register, all hospitalizations for UGIBs were identified among the 156 138 users of NSAIDs and compared with the number of expected based on the North Jutland population who did not receive NSAID prescriptions.

Results  During periods of NSAID use without use of other drugs associated with UGIB, we observed 365 UGIBs, a number 3.6 times higher than expected (95% CI = 3.3, 4.0). The excess risk varied by sex, type of NSAID and form and route of administration of the NSAID, but not by age at first NSAID prescription or number of prior prescriptions. Risk declined sharply following cessation of use. For ibuprofen and naproxen, there was a clear trend in rising risk by increasing dose, although the lowest doses were also associated with an excess of UGIB. Concurrent use of corticosteroids, anticoagulants and aspirin further increased the risk of UGIB.

Conclusions  All types and formulations of NSAIDs appear to increase the risk of UGIBs, but the effect appear not to be cumulative and diminish rapidly with discontinue of use. Up to 15% of the UGIBs in the entire population of the North Jutland County may be explained by use of this drug.
Stopping rules for phase II studies
Authors: Stallard Nigel , Whitehead John , Todd Susan , Whitehead Anne
Published: March 2003

This paper, the second in a series of three papers concerned with the statistical aspects of interim analyses in clinical trials, is concerned with stopping rules in phase II clinical trials. Phase II trials are generally small‐scale studies, and may include one or more experimental treatments with or without a control. A common feature is that the results primarily determine the course of further clinical evaluation of a treatment rather than providing definitive evidence of treatment efficacy. This means that there is more flexibility available in the design and analysis of such studies than in phase III trials. This has led to a range of different approaches being taken to the statistical design of stopping rules for such trials. This paper briefly describes and compares the different approaches. In most cases the stopping rules can be described and implemented easily without knowledge of the detailed statistical and computational methods used to obtain the rules.
Diurnal variation in the biliary excretion of flomoxef in patients with percutaneous transhepatic biliary drainage
Authors: Hishikawa Shuji , Kobayashi Eiji , Sugimoto Koh‐ichi , Miyata Michio , Fujimura Akio
Published: March 2003

Aims  To examine diurnal variation in biliary excretion of flomoxef.

Methods  Flomoxef (1 g) was injected intravenously in eight patients with percutaneous transhepatic cholangiography with drainage at 09.00 h and 21.00 h by a cross‐over design with a 36 h washout period. Drained biliary fluid was collected for 6 h after each dosing. These patients still had mild to moderate hepatic dysfunction.

Results  Bile flow and bile acid excretion for 6 h after dosing did not differ significantly between the 09.00 h and 21.00 h treatments. The maximum concentration of biliary flomoxef was significantly greater and its total excretion for 6 h tended to be greater after the 21.00 h dose [maximum concentration (µg ml−1): 34.2 ± 29.9 (09.00 h dose) vs 43.5 ± 28.3 (21.00 h dose) (95% confidence interval for difference: 2.6∼15.9, P = 0.013); total excretion (mg 6 h−1): 1.4 ± 1.3 (09.00 h dose) vs 1.6 ± 1.2 (21.00 h dose) (95% confidence interval for difference: −26.8, 313.7, P = 0.087)]. The period that biliary flomoxef remained above the minimal inhibitory concentration did not differ significantly between the two treatment times.

Conclusions  These results suggest that biliary excretion of flomoxef shows diurnal variation. However, as the difference was relatively small, flomoxef could be given at any time of day without any dosage adjustments.
A new laser pain threshold model detects a faster onset of action from a liquid formulation of 1 g paracetamol than an equivalent tablet formulation
Authors: Sutton J. A. , Gillin W. P. , Grattan T. J. , Clarke G. D. , Kilminster S. G.
Published: March 2003

Aims  To discover whether a new infra‐red laser method could detect a change in pain threshold after as mild an analgesic as paracetamol and whether an effervescent liquid formulation produced a faster onset of action than tablets.

Methods 
This double‐blind, placebo controlled randomized study used a portable, infra‐red laser to measure ‘first pain’ thresholds on the nondominant forearm in 12 normal volunteers before and after 1 g of paracetamol or placebo. The mean of six recordings was determined three times before dosing, the first being used as a familiarization procedure, and 14 times after dosing.

Results 
We detected a small (2%), statistically significant difference in pain threshold between a liquid formulation of paracetamol and placebo at 30 and 60 min (P = 0.004 and P = 0.001), but not between tablets and placebo. Liquid also increased the threshold significantly compared with tablets at 60 min (P = 0.01).

Conclusions To detect such a small increase in pain threshold requires a highly consistent measure and the coefficient of variation was 2% for the study overall, surprisingly low for a subjective phenomenon. The reasons for this include minimizing reflectance by blacking the skin, using a nonhairy site, averaging six data points at each sample time and controlling closely the ambient conditions and the subjects' preparation for studies.
Sex and age differences in the pharmacokinetics of alosetron
Authors: Koch K. M. , Palmer J. L. , Noordin N. , Tomlinson J. J. , Baidoo C.
Published: March 2003

Aims  To determine the effects of sex and age on the pharmacokinetics of alosetron.

Methods  Single oral and intravenous 2 mg doses of alosetron were administered on separate occasions to 48 healthy, young and elderly, males and females. Serum was sampled for 12 h post‐dose to measure alosetron concentrations.

Results  Serum concentrations of alosetron were higher in females than in males, resulting from a sex difference in clearance by metabolism. Mean clearance values were 504 vs 677 ml min−1 in young females vs males (mean ratio 0.75), and 461 vs 670 ml min−1 in elderly females vs males (mean ratio 0.69). The sex difference in alosetron pharmacokinetics achieved statistical significance in the elderly, but not in the young. Irrespective of sex, alosetron clearance was increased by smoking. Serum concentrations tended to be higher in the elderly, although the effect of age was generally not significant. Volume of distribution was smaller in females (approximately 63 l) compared with males (approximately 84 l), regardless of age or the sex difference in body weight.

Conclusions  A significant difference in clearance by metabolism of alosetron between the sexes, and possibly between the young and elderly was observed.
A uniform regimen enables decentralized care of diabetic pregnancies
Authors: Vääräsmäki M. , Gissler M. , Hartikainen A.‐L.
Published: March 2003

Aims  A nationwide recommendation to standardize the care of diabetic pregnancies in different hospitals was given in Finland in 1993. The Medical Birth Register (MBR) was used to investigate whether these recommendations have been accepted and how they have affected the outcome of newborns.

Methods  Data on 1442 singleton pregnancies complicated by insulin‐treated diabetes in 1991–1995 were obtained from the MBR.

Results  The incidence of insulin treatment during pregnancy was 4.5 per 1000 births. Sixty‐six per cent (n = 954) of all women had Type 1 diabetes. During the study period, the number of deliveries managed in tertiary centres decreased from 59% to 47% (95% confidence interval [CI] 39–58%) and care was more often carried out on an out‐patient basis. The perinatal mortality rate (≥ 28 weeks of gestation) declined from 19.3 to 8.2/1000, being 12.6/1000 in the whole diabetic population and 5.5/1000 in the general population (95% CI 3.4–8.8/1000). The risk was especially increased in insulin‐treated gestational diabetic (GDM) pregnancies (14.3/1000). The proportion of macrosomic newborns (31.7%) in diabetic women was significantly higher than among the general population (3.2%) (95% CI 27.0–33.9%).

Conclusions  The decentralization and change‐over to a mainly out‐patient basis of management does not appear to have increased the number of cases of adverse outcome of diabetic pregnancy when patients have been selected to the appropriate level of care. The risks in insulin‐treated GDM pregnancies were almost similar to Type 1 diabetes. To succeed, there must be a standardized care programme, continuous education and motivated personnel.

Diabet. Med. 18, 871–876 (2001)
Do levels of immunoglobulin G antibodies to foods predict the development of immunoglobulin E antibodies to cat, dog and/or mite?
Authors: Eysink P. E. D. , Bindels P. J. E. , Stapel S. O. , Bottema B. J. A. M. , Van Der Zee J. S. , Aalberse R. C.
Published: March 2003

Background In children at high risk of inhalation allergy, food sensitization is associated with an increased risk for sensitization to inhalant allergens. Furthermore, this association was also found in a cross‐sectional study.

Objective To examine in a prospective study, whether levels of IgG to foods (i.e. mixture of wheat and rice, mixture of soy bean and peanut, egg white, cow's milk, meat, orange and potato) indicate an increased risk for the future development of IgE antibodies to inhalant allergens in a low‐risk population and whether they can be used as predictors of the subsequent development of IgE antibodies in young, initially IgE‐negative children.

Methods Coughing children, aged 1–5, visiting their GPs, were tested for IgE antibodies to mite, dog and cat (RAST) and IgG (ELISA) to foods. All IgE‐negative children were retested for IgE antibodies after two years. The IgG results (66 percentiles) of the first blood sample were compared to the RAST‐scores of the second blood sample.

Results After two years, 51 out of 397 (12.8%) originally IgE‐negative children, had become IgE‐positive for cat, dog and/or mite. An increased IgG antibody level to wheat‐rice (OR = 2.2) and to orange (OR = 2.0) indicated an increased risk of developing IgE to cat, dog or mite allergens. In addition to IgG to a mixture of wheat‐rice and orange; total IgE, breastfeeding, eczema as a baby and age were the most important predictors for the subsequent development of IgE to inhalant allergens.

Discussion An increased IgG antibody level to a mixture of wheat‐rice or orange, indicates an increased risk of developing IgE to cat, dog or mite allergens. This indicates that excessive activity of the mucosal immune system is present before IgE antibodies to airborne allergens can be demonstrated. Nevertheless, IgG to foods is not very helpful (with a positive predictive value of 16.5%, and negative predictive value of 90.6%) in identifying individual children at risk in clinical practice. However, besides other risk factors, IgG to wheat‐rice and to orange could be useful as a screening test for studies in the early identification, i.e. before IgE antibodies can be detected, of children with an increased risk of developing IgE antibodies in the future.
Specific monoclonal antibodies and human immunoglobulin E show that Hev b 5 is an abundant allergen in high protein powdered latex gloves
Authors: Sutherland M. F. , Drew A. , Rolland J. M. , Slater J. E. , Suphioglu C. , O'Hehir R. E.
Published: March 2003

Background  Hev b 5 is a major latex allergen recognized predominantly by latex‐allergic health care workers (HCWs). Recombinant Hev b 5 (rHev b 5) was previously expressed as a fusion protein with maltose binding protein (MBP), itself an immunogenic molecule; therefore non‐fusion rHev b 5 is desirable. Moreover, standardized immunological assays for the detection of Hev b 5 are currently lacking and may have important implications for both allergen avoidance and diagnosis in latex allergy.

Objectives  To generate and use Hev b 5‐specific mAbs to determine the relative abundance of Hev b 5 in different latex extracts, correlating this with the IgE reactivity of latex‐allergic HCWs and to produce non‐fusion rHev b 5.

Methods  For the production of mAbs, mice were immunized with rHev b 5/MBP fusion protein and mAbs selected with rHev b 5/MBP but not MBP reactivity. The mAb reactivity was compared with polyclonal IgE from latex‐allergic HCWs using direct and inhibition ELISA and immunoblot assays. Recombinant Hev b 5 was expressed and purified in the pPROEX‐HTa bacterial expression system.

Results  Four Hev b 5‐specific mAbs were produced. Immunoblotting and ELISA using the mAbs indicate abundant Hev b 5 in high protein powdered latex glove extracts as compared with crude latex sap extracts. High quality surgical gloves with no detectable protein have no detectable Hev b 5. Inhibition ELISAs using serum IgE from latex‐allergic HCWs and Hev b 5‐specific mAbs gave strong correlation. Non‐fusion recombinant Hev b 5 was successfully expressed and purified, showing reactivity with both the Hev b 5‐specific mAbs and serum IgE of latex‐allergic HCWs.

Conclusion  Hev b 5‐specific mAbs and human IgE from latex‐allergic HCWs demonstrate the greater content of Hev b 5 in high protein powdered glove extracts. This may explain the observed higher frequency of sensitization to this allergen in HCWs.
The human complement C9 gene: structural analysis of the 5′ gene region and genetic polymorphism studies
Authors: Witzel‐Schlömp K. , Rittner C. , Schneider P. M.
Published: March 2003

C9 is the last of the human complement components creating the membrane attack complex. The single chain serum protein is encoded by a gene located on chromosome 5p13 that is composed of 11 exons. With the aid of inverse PCR, the hitherto unknown regions flanking exon 1 and the 3′ part of exon 11 (3′UTR) have been sequenced. A computer‐based analysis of the 300‐bp region located just upstream of the AUG start codon showed homologies to known DNA modules which affect the transcriptional regulation of certain genes. The most striking of these is a sequence that may substitute the missing TATA box in initiating C9 transcription. In the 3′UTR, three successive polyadenylation signals were found. Although the C9 protein is invariant, four different single nucleotide polymorphisms (SNPs) have been observed at the DNA level by exon‐specific PCR and direct sequencing. None of them changes the amino acid composition of the mature protein. Due to a C → T transition in exon 1 at cDNA position 17, the fifth amino acid of the leader peptide may be either an arginine or a tryptophane. Using either PCR/RFLP analysis (exons 1 and 11) or allele‐specific PCR (intron 1 and exon 4), each polymorphism can be characterized without sequencing. All of the exon 1, intron 1 and exon 11 variants could be detected in small population samples of European, Thai or South American Indian origin. In contrast, the exon 4 C variant was observed only once in a European. The first three SNPs can be combined to designate eight different ‘C9 alleles’. Of these, six have actually be found. These data provide strong evidence that several mutation and recombination events occurred in the course of C9 gene evolution.
Polymorphisms of tumour necrosis factors A and B in breast cancer
Authors: Park K. S. , Mok J. W. , Ko H. E. , Tokunaga K. , Lee M. H.
Published: March 2003

We assayed for germline single nucleotide polymorphisms (SNPs) in theTNFBandTNFAgenes in patients with breast cancer. SNPs were observed in the first intron ofTNFB(G/A) and at −1031 (T/C), −863 (C/A), −857 (C/T) and −308 (G/A) in the promoter region ofTNFAfrom peripheral leucocytes in 95 breast cancer patients and 190 healthy subjects as controls. TheTNFB*G/TNFB*Ghomozygote (23.2% vs. 5.8%,P= 0.001) was predominant in patients, while theTNFB*A/TNFB*Ahomozygote was less frequent in patients (34.7% vs. 46.3%,P = 0.041) than in the control subjects. Breast cancer was not associated with SNPs in theTNFApromoters. Although theTNFBSNP failed to associate with any clinicopathological parameter of breast cancer, a substantial difference in pathology among tumour stages for the −857 SNP inTNFAwas detected. These results indicate thatTNFBhas both tumorigenic and antitumorigenic capabilities depending on the genotype: theTNFBSNPTNFB*G/TNFB*Ggenotype gave an increased risk for breast cancer and that ofTNFB*A/TNFB*Agave resistance to breast cancer (OR = 5.3395%; CI: 2.33–12.19). The results suggest that theTNFB*Gallele plays some role in the tumorigenesis or activation of dormant tumour cells, but theTNFB*Aallele induces some function(s) leading to the inhibition of tumorigenesis.
Molecular analysis of HLA‐DRB1 polymorphism in north‐east Spain
Authors: Vidal S. , Morante M. P. , Moga E. , Mosquera A. M. , Querol S. , Garcia J. , Rodriguez‐Sanchez J. l.
Published: March 2003

The DRB1* polymorphism in 941 randomly selected individuals from the Umbilical Cord Blood Bank of Barcelona (92.75% of Spanish origin) was determined by sequence‐based typing. The HLA profile was similar to that of other Mediterranean populations, with DRB1*0701 and *0301 being the most frequent alleles. This may be a consequence of the mixture of alleles as a result of migration from contiguous geographical areas.
What is the correct primary diagnostic tool in the ‘one‐stop’ clinic for the investigation of abnormal menstrual bleeding?
Authors: Jones Kevin D. , Jermy Karen , Bourne Tom H.
Published: March 2003

Objective To review the management in the ‘one‐stop’ clinic of abnormal menstrual bleeding.

Design Review.

Results The combination of transvaginal ultrasonography (TVS) with s‐HS (saline hydrosonography) has made ultrasound without hysteroscopy a potential primary diagnostic tool in the one‐stop abnormal menstrual bleeding clinic. This is particularly true if outpatient hysteroscopy is carried out with blind endometrial sampling, rather than a directed biopsy. In the detection of focal endometrial pathology, compared with TVS alone, TVS plus s‐HS increases the sensitivity from 67% to 87%, the specificity from 89% to 91%, the positive predictive value from 88% to 92% and the negative predictive value from 71% to 86%. Furthermore, TVS facilitates the diagnosis of adnexal and intramural pathology.

Conclusion We believe that a critical appraisal of the evidence supports TVS as the correct primary diagnostic tool in the evaluation of abnormal menstrual bleeding in a one‐stop clinic.
Metal fume fever: A case report and literature review
Authors: Merchant John , Webby Rosalind
Published: March 2003

Metal fume fever is a relatively uncommon presentation to the emergency department. Resulting from inhalation of metal oxides, it presents as an acute, self‐limiting, flu‐like symptom complex often misdiagnosed as a viral illness. We report a case of a 26‐year‐old male presenting with metal fume fever 4 h after oxycutting galvanized steel. The literature is reviewed with respect to pathogenesis, the common presenting symptom complex, investigations and current accepted management of metal fume fever.