bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus
Authors: De Smet Lina , Pettigrew Graham W. , Van Beeumen Jozef J.
Published: March 2003

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over‐expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7‐kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21‐amino‐acid N‐terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico‐chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high‐potential (Em=+270 mV) and a low‐potential haem (between −190 mV and −310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high‐spin state at the low‐potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities (‘turnover number’) are 85 800·min−1 and 63 600·min−1, respectively. These activities are the highest ever found for a BCCP.
Functional expression of human liver cytosolic β‐glucosidase in Pichia pastoris
Authors: Berrin Jean‐Guy , McLauchlan W. Russell , Needs Paul , Williamson Gary , Puigserver Antoine , Kroon Paul A. , Juge Nathalie
Published: March 2003

Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic β‐glucosidase (CBG) that hydrolyses various β‐d‐glycosides, but whose physiological function is not known. Here, we describe the first heterologous expression of human CBG, a system that facilitated␣a detailed assessment of the enzyme specificity towards dietary glycosides. A full‐length CBG cDNA (cbg‐1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of ≈ 10 mg·L−1. The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4‐nitrophenyl‐β‐d‐glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl‐glycosides (β‐d‐fucosides, α‐l‐arabinosides, β‐d‐glucosides, β‐d‐galactosides, β‐l‐xylosides, β‐d‐arabinosides), similar to the native enzyme. For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (Km) and specificities (kcat/Km) for dietary xenobiotics than for other aryl‐glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.
Functional assignment of motifs conserved in β1,3‐glycosyltransferases
Authors: Malissard Martine , Dinter André , Berger Eric G. , Hennet Thierry
Published: March 2003

The β1,3‐glycosyltransferase enzymes identified to date share several conserved regions and conserved cysteine residues, all being located in the putative catalytic domain. To investigate the importance of these motifs and cysteines for the enzymatic activity, 14 mutants of the murine β1,3‐galactosyltransferase‐I gene were constructed and expressed in Sf9 insect cells. Seven mutations abolished the galactosyltransferase activity. Kinetic analysis of the other seven active mutants revealed that three of them showed a threefold to 21‐fold higher apparent Km with regard to the donor substrate UDP‐galactose relative to the wild‐type enzyme, while two mutants had a sixfold to 7.5‐fold increase of the apparent Km value for the acceptor substrate N‐acetylglucosamine‐β‐p‐nitrophenol. Taken together, our results indicate that the conserved residues W101 and W162 are involved in the binding of the UDP‐galactose donor, the residue W315 in the binding of the N‐acetylglucosamine‐β‐p‐nitrophenol acceptor, and the domain including E264 appears to participate in the binding of both substrates.
Implications of the simultaneous occurrence of hepatic glycolysis from glucose and gluconeogenesis from glycerol
Authors: Phillips John W. , Jones Michael E. , Berry Michael N.
Published: March 2003

Glycolysis from [6‐3H]glucose and gluconeogenesis from [U‐14C]glycerol were examined in isolated hepatocytes from fasted rats. A 5 mm bolus of glycerol inhibited phosphorylation of 40 mm glucose by 50% and glycolysis by more than 60%, and caused cellular ATP depletion and glycerol 3‐phosphate accumulation. Gluconeogenesis from 5 mm glycerol was unaffected by the presence of 40 mm glucose. When nonsaturating concentrations of glycerol (< 200 µm) were maintained in the medium by infusion of glycerol, cellular ATP concentrations remained normal. The rate of uptake of infused glycerol was unaffected by 40 mm glucose, but carbohydrate synthesis from glycerol was inhibited 25%, a corresponding amount of glycerol being diverted to glycolytic products, whereas 10 mm glucose had no inhibitory effect on conversion of infused glycerol into carbohydrate. Glycerol infusion depressed glycolysis from 10 mm and 40 mm glucose by 15 and 25%, respectively; however, the overall rates of glycolysis were unchanged because of a concomitant increase in glycolysis from the infused glycerol.

These studies show that exposure of hepatocytes to glucose and low quasi‐steady‐state concentrations of glycerol result in the simultaneous occurrence, at substantial rates, of glycolysis from glucose and gluconeogenesis from the added glycerol. We interpret our results as demonstrating that, in hepatocytes from normal rats, segments of the pathways of glycolysis from glucose and gluconeogenesis from glycerol are compartmentalized and that this segregation prevents substantial cross‐over of phosphorylated intermediates from one pathway to the other. The competition between glucose and glycerol implies that glycolysis and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis may indicate channelling within the cytoplasm of individual hepatocytes.
Lipid rafts and little caves
Authors: Zajchowski Laura D. , Robbins Stephen M.
Published: March 2003

Lipid rafts are liquid‐ordered membrane microdomains with a unique protein and lipid composition found on the plasma membrane of most, if not all, mammalian cells. A large number of signalling molecules are concentrated within rafts, which have been proposed to function as signalling centres capable of facilitating efficient and specific signal transduction. This review summarizes current knowledge regarding the composition, structure, and dynamic nature of lipid rafts, as well as a number of different signalling pathways that are compartmentalized within these microdomains. Potential mechanisms through which lipid rafts carry out their specialized role in signalling are discussed in light of recent experimental evidence.
Characteristics of binding of insulin‐like growth factor (IGF)‐I and IGF‐II analogues to the type 1 IGF receptor determined by BIAcore analysis
Authors: Forbes Briony E. , Hartfield Perry J. , McNeil Kerrie A. , Surinya Kathy H. , Milner Steven J. , Cosgrove Leah J. , Wallace John C.
Published: March 2003

Insulin‐like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF‐I, IGF‐II and 14 IGF analogues to a recombinant high‐affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF‐binding protein, which exhibited IGF1R‐binding affinities similar to those of IGF‐I or IGF‐II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R‐binding affinities. The relative binding affinities of IGF‐I analogues and IGF‐I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor‐binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R‐binding affinities were related to ability to protect against serum withdrawal‐induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF‐I and IGF‐II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.
The nuclear genome is involved in heteroplasmy control in a mitochondrial mutant strain of Drosophila subobscura
Authors: Farge Géraldine , Touraille Sylvie , Le Goff Samuel , Petit Nathalie , Renoux Monique , Morel Frédéric , Alziari Serge
Published: March 2003

Most (78%) mitochondrial genomes in the studied mutant strain of Drosophila subobscura have undergone a large‐scale deletion (5 kb) in the coding region. This mutation is stable, and is transmitted intact to the offspring. This animal model of major rearrangements of mitochondrial genomes can be used to analyse the involvement of the nuclear genome in the production and maintenance of these rearrangements. Successive backcrosses between mutant strain females and wild‐type males yield a biphasic change in heteroplasmy level: (a) a 5% decrease in mutated genomes per generation (from 78 to 55%), until the nuclear genome is virtually replaced by the wild‐type genome (seven to eight crosses); and (b) a continuous decrease of 0.5% per generation when the nuclear context is completely wild‐type. In parallel with these changes, NADH dehydrogenase activity, which is halved in the mutant strain (five subunits of this complex are affected by the mutation), gradually increases and stabilizes near the wild‐type activity. A return to a nuclear context is accompanied by the opposite phenomena: progressive increase in heteroplasmy level and stabilization at the value seen in the wild‐type strain and a decrease in the activity of complex I. These results indicate that the nuclear genome plays an important role in the control of heteroplasmy level and probably in the production of rearranged genomes.
Expression of V‐ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3‐deficient strain of Saccharomyces cerevisiae and its complementation study
Authors: Ikeda Mikiko , Hinohara Misato , Umami Kimiko , Taguro Yuki , Okada Yoshio , Wada Yoh , Nakanishi Yoichi , Maeshima Masayoshi
Published: March 2003

The function of the translation products of six different cDNAs for Acetabularia V‐ATPase proteolipid subunit (AACEVAPD1 to AACEVAPD6) was examined using a Saccharomyces cerevisiae VMA3‐deficient strain that lacked its own gene for one of the proteolipid subunits of V‐ATPase. Expression of the cDNAs in the strain revealed that four cDNAs from the six complemented the proton transport activity into the vacuole, visualized by fluorescence microscopy. The vacuolar‐membrane‐enriched fractions from the four transformants showed cross‐reactivity with antibodies against the subunits a and A of S. cerevisiae V‐ATPase. Two translation products from the other two cDNAs were demonstrated not to be localized in vacuolar membranes, and thus could not complement the function of the VMA3‐deficient strain. As the primary structures deduced from the former four cDNAs are similar but clearly different from those of the latter two, the latter two translation products may not be able to substitute for theVMA3 gene product.
Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli
Authors: Weiß H. Markus , Grisshammer Reinhard
Published: March 2003

The adenosine A2a receptor belongs to the seven transmembrane helix G‐protein‐coupled receptor family, is abundant in striatum, vasculature and platelets and is involved in several physiological processes such as blood pressure regulation and protection of cells during anoxia. For structural and biophysical studies we have expressed the human adenosine A2a receptor (hA2aR) at high levels inserted into the Escherichia coli inner membrane, and established a purification scheme. Expression was in fusion with the periplasmic maltose‐binding protein to levels of 10–20 nmol of receptor per L of culture, as detected with the specific antagonist ligand [3H]ZM241385. As the receptor C‐terminus was proteolyzed upon solubilization, a protease‐resistant but still functional receptor was created by truncation to Ala316. Addition of the sterol, cholesteryl hemisuccinate, allowed a stable preparation of functional hA2aR solubilized in dodecylmaltoside to be obtained, and, increased the stability of the receptor solubilized in other alkylmaltosides. Purification to homogeneity was achieved in three steps, including ligand affinity chromatography based on the antagonist xanthine amine congener. The purified hA2aR fusion protein bound [3H]ZM241385 with a Kd of 0.19 nm and an average Bmax of 13.7 nmol·mg−1 that suggests 100% functionality. Agonist affinities for the purified solubilized receptor were higher than those for the membrane‐bound form. Sufficient pure, functional hA2aR can now be prepared regularly for structural studies.
Engineering and use of 32P‐labeled human recombinant interleukin‐11 for receptor binding studies
Authors: Wang Xiao‐Ming , Wilkin Jean‐Marc , Boisteau Olivier , Harmegnies Dimitri , Blanc Chrystel , Vandenbussche Paul , Montero‐Julian Félix A. , Jacques Yannick , Content Jean
Published: March 2003

Human interleukin‐11 (hIL‐11) is a pleiotropic cytokine that is involved in numerous biological activities such as hematopoiesis, osteoclastogenesis, neurogenesis and female fertility. IL‐11 is obviously a key reagent to study the IL‐11 receptors. However, conventional radio‐iodination techniques lead to a loss of IL‐11 bioactivity. Here, we report the construction and the production of a new recombinant human IL‐11 (FPΔIL‐11). In this molecule, a specific phosphorylation site (RRASVA) has been introduced at the N‐terminus of rhIL‐11. It can be specifically phosphorylated by bovine heart protein kinase and accordingly, easily radiolabeled with 32P. A high radiological specific activity (250 000 c.p.m.·ng−1 of protein) was obtained with the retention of full biological activity of the protein. The binding of 32P‐labeled FPΔIL‐11 to Ba/F3 cells stably transfected with plasmids encoding human IL‐11 receptors α and β chains (IL‐11Rα and gp130) was specific and saturable with a high affinity as determined from Scatchard plot analysis. Availability of this new ligand should prompt further studies on IL‐11R structure, expression and regulation.
Coordinated action of protein tyrosine phosphatases in insulin signal transduction
Authors: Cheng Alan , Dubé Nadia , Gu Feng , Tremblay Michel L.
Published: March 2003

Insulin is the principal regulatory hormone involved in the tight regulation of fuel metabolism. In response to blood glucose levels, it is secreted by the β cells of the pancreas and exerts its effects by binding to cell surface receptors that are present on virtually all cell types and tissues. In humans, perturbations in insulin function and/or secretion lead to diabetes mellitus, a severe disorder primarily characterized by an inability to maintain blood glucose homeostasis. Furthermore, it is estimated that 90–95% of diabetic patients exhibit resistance to insulin action. Thus an understanding of insulin signal transduction and insulin resistance at the molecular level is crucial to the understanding of the␣pathogenesis of this disease. The insulin receptor (IR) is a transmembrane tyrosine kinase that becomes activated upon ligand binding. Consequently, the receptor and its downstream substrates become tyrosine phosphorylated. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate biological response. One important mechanism by which insulin signaling is regulated involves the protein tyrosine phosphatases (PTPs), which may either act on the IR itself and/or its substrates. Two well characterized examples include leuckocyte antigen related (LAR) and protein tyrosine phosphatase‐1B (PTP‐1B). The present review will discuss the current knowledge of these two and other potential PTPs involved in the insulin signaling pathway.
Drought mortality of bush elephants in Hwange National Park, Zimbabwe
Authors: Dudley J. P. , Criag G. C. , Gibson D. ST. C. , Haynes G. , Klimowicz J.
Published: March 2003

African bush elephants inhabiting the undeveloped Kalahari Sands region of Hwange National Park, Zimbabwe are subject to episodic mortality during droughts. We monitored the drought‐related mortality of elephants in Hwange National Park over the course of an extended drought between 1993 and 1995. The drought‐related mortality of elephants was higher during 1994 than 1995, despite significantly higher rainfall in 1994 than 1995. We found significant differences in the age‐specific mortality of elephants during 1994 and 1995. The cumulative mortality profile from this study differed significantly from previous die‐offs at this site, with a higher mortality among adult age classes than that reported from earlier studies in Hwange National Park. The effective duration of the rainy season, not total annual precipitation, appears to be the best predictor for the potential severity of drought mortality among elephants in the Kalahari Sands habitats of Hwange National Park.
Gene therapy for the treatment of disorders of the musculo‐skeletal system
Authors: Goomer R.
Published: March 2003

Mimicking a developmental gene expression pathway may be the best means for repairing or healing orthopedic tissues. Identification and isolation of genes that are critical inducers of these regulatory pathways is a significant first step towards this goal. Delivering the genes of interest into the appropriate environment with the least amount of side‐effects is crucial.

Successful in vivo gene delivery requires development of methodologies that are highly efficient and would not induce a host immune response. I will discuss a non‐viral gene delivery that we have used to deliver genes with high efficiency in a cartilage defect repair model in the rabbit and for direct delivery of the marker gene into canine flexor tendons during repair surgery. Using this gene delivery approach, efficiencies exceeding 70% have been demonstrated in both models. Specific growth and transcription factors are now being used in this gene therapy system.

In addition to the specific growth and regulatory transcription factors, subtractive hybridization or differential gene expression profiling is identifying critical new genes involved in the healing process. I will present one example where we have identified a ras activated, proliferation dependent PT‐12 gene. PT‐12 is expressed by patellar tendon, a tissue that heals readily, but is completely absent in anterior cruciate ligament, a tissue that fails to raise a successful repair response following injury. I will discuss the potential role of this as a marker of tissue repair and healing.

Finally, the potential for specific morphoregulatory factors such as growth and transcription factors and adhesion molecules in cartilage repair and ligament healing and antifibrosis in gene therapy will be discussed.
Banded iron formations and palaeoenvironment: a problem in petrogenesis
Authors: Page David
Published: March 2003

The mineralogy, chemistry and stratigraphy of Precambrian banded iron formations have been extensively documented, yet the way such formations are produced remains a subject for debate. Differentiation by varving, microbial precipitation and secondary alteration are all seen as possible mechanisms, but discussion returns to the lack of any modern analogue. Nothing like banded iron forms anywhere in the world today or has done during the entire Phanerozoic. Where do we begin with such enigmatic rocks?
Exercise training lowers the resting metabolic rate of Zebra Finches, Taeniopygia guttata
Authors: Nudds R. L. , Bryant D. M.
Published: March 2003

Although changes in the mass and biochemistry of vertebrate locomotory muscle are established responses to exercise training, the effects of training on resting metabolic rate (RMR) are not clear. Here, a sustained flight training regime was shown to lower both the night‐time and day RMR of captive Zebra Finches (Taeniopygia guttata). Training also reduced fat reserves and overall body mass.

 Our results are largely consistent with the effects of an elevated energy expenditure on night‐time RMR detected in a study of workload on Zebra Finches but contrast with most studies of humans, where exercise training increases or has no effect upon RMR.

 A compensatory lowering of RMR in response to increased physical activity may be an artefact of captivity and could be dependent upon the activity levels of the captives concerned.

 It is suggested that when extrapolating from cage bird studies to wild situations, consideration should be given to the physical condition of the caged individuals being used.

 Variation in physical fitness may account for some of the unexplained variation in comparative studies of basal metabolism.
Massive use of chemotherapy influences life traits of parasitic nematodes in domestic ruminants
Authors: Leignel V. , Cabaret J.
Published: March 2003

The size of nematodes is an operational indicator of fecundity. The variation in size due to chemotherapy was studied with a benzimidazole anthelmintic in susceptible and resistant nematodes.Teladorsagia circumcincta circumcincta(Stadelmann 1894), a nematode endoparasite of sheep and goats, was investigated as many components of its fitness are already known.

 Susceptible worms submitted to increasing selective pressure by anthelmintics increased in size (by 6–10%); it was hypothesized that this was partly under the control of sheep, as treated lambs may mount and maintain a better response when infected (premunition).

 The resistant worms, whatever the anthelmintic pressure, were always 3% larger than susceptible ones.

 Thus, size may increase in susceptible worms and resistant worms for very different reasons. If resistance does not emerge, massive chemotherapy should lead to larger nematodes, and hence to more fertile worms.
The influence of propagule size and maternal nest‐site selection on survival and behaviour of neonate turtles
Authors: KOLBE J. J. , JANZEN F. J.
Published: March 2003

Maternal effects are widely recognized as important ecological and evolutionary forces, with fitness consequences for both offspring and parents. However, little is known about the impact of maternal effects on the survival and behaviour of offspring in the field. Two potentially important maternal effects that influence the phenotypes and survival of neonates are (i) propagule size effects (i.e. egg size, hatchling size and clutch effects) and (ii) nest‐site selection, which influences incubation conditions and the suitability of habitat for neonates after hatching.

 We experimentally removed the effects of variable incubation conditions to focus on how propagule size effects and nest‐site microhabitat characteristics influence hatchling survival and behaviour in the Common Snapping Turtle,Chelydra serpentina. Using eight simultaneous experimental releases of neonates under ecologically relevant field conditions, we detected no influence of propagule size effects on hatchling turtle survival or behaviour. Egg mass, hatchling mass, hatchling carapace length and clutch did not predict the probability of survival, nor did they influence hatchling behaviour.

 However, nest‐site microhabitat characteristics were important for both survival and behaviour. The probability of hatchling survival increased with decreasing amounts of ground vegetation and slope at the release point. Hatchlings took longer to reach the fence when released in areas with more ground vegetation and dispersed farther when released from greater distances and at lower slopes. Therefore, variation in maternal behaviour, such as nest‐site selection, is an important effect because it results in differential survival and behaviour of offspring in this population, whereas variation in maternal size effects alone does not.
Limits to food intake by the Prairie Vole: effects of time for digestion
Authors: Zynel C. A. , Wunder B. A.
Published: March 2003

Prairie Voles (Microtus ochrogaster Wagner) are among the smallest vegetation‐feeding mammalian herbivores, and thus show a high ratio of energetic expenditure to energy content of ingested food that may lead to digestive bottlenecks. By varying the periodicity of 3 h of food availability, it was found that energy assimilation in Prairie Voles is not limited by the time required to consume food, but by the time required to digest and absorb food.

Three hours is all the time that is necessary for a Prairie Vole to consume the food needed for 1 day. Although voles offered food in one 3‐h block per 24 h increased dry matter digestibility, they could not consume enough food to maintain body mass. However, when offered 3 h of food availability but divided into six 30‐min periods evenly spaced throughout 24 h Prairie Voles reached a level of food intake similar to that of control voles fed ad libitum and stabilized body mass similar to controls.

It is suggested that a vole’s stomach capacity limits intake for each feeding bout, regardless of how empty the following chambers are after any period of food restriction and the rate of stomach emptying may dictate the feeding bout periodicity. The amount of stomach contents (1·1–1·4 g) found in voles held under similar conditions to those in our experiments closely resembles the amount of food consumed (0·78–1·4 g) during each of the six 30‐min periods of feeding.

The bottleneck limits intake of food and keeps feeding bouts spaced. The time required for digestion may be the cause for a vole’s observed 2–4 h ultradian activity rhythm. Evidence is given that spacing between activity periods may be adjusted to the metabolic demands of the species.
Juvenile Atlantic Salmon (Salmo salar) with relatively high standard metabolic rates have small metabolic scopes
Authors: Cutts C. J. , Metcalfe N. B. , Taylor A. C.
Published: March 2003

There is an established relationship between metabolism and behavioural dominance in several fish species, such that individuals with higher standard metabolic rates tend to be more dominant. It has also been suggested that metabolic scope increases with standard metabolic rate.

  Energetically expensive behaviours attributable to dominance, such as aggression, can be carried out to a greater extent within larger metabolic scopes. This study therefore tests the hypothesis that juvenile salmon with higher standard metabolic rates (which tend to be dominant) have larger metabolic scopes.

  It was found that salmon with high standard metabolic rates (corrected for body mass) in fact had smaller metabolic scopes than conspecifics with relatively low standard metabolic rates. Possible reasons for their potential dominance while having small metabolic scopes and high relative standard metabolic rates are discussed.
Multigenerational familial medullary thyroid cancer (FMTC): evidence for FMTC phenocopies and association with papillary thyroid cancer
Authors: Fugazzola L. , Cerutti N. , Mannavola D. , Ghilardi G. , Alberti L. , Romoli R. , Beck‐Peccoz P.
Published: March 2003

background Occurrence in a familial setting is well established for medullary thyroid carcinoma (MTC) and has been more recently reported for papillary thyroid cancer (PTC). Germline mutations or rearrangements of the RET proto‐oncogene are the genetic background of the majority of hereditary MTCs and of about 25–40% of PTCs.

patients A large multigenerational familial medullary thyroid cancer (FMTC) family, comprised of four generations and a total of 60 subjects, has been fully evaluated. Studies on germline RET mutations and polymorphisms, on somatic RET activation and on haplotyping with RET‐linked markers, were performed.

results RET mutational analysis revealed a rare missense point mutation in exon 15 of RET (A891S), associated with FMTC. Haplotype analysis showed a co‐segregation between the allelic variant 5 of D10S578 marker (which is tightly linked to the RET locus) and the RET mutation. Two patients, from different branches of the family, did not harbour the point mutation A891S despite histological confirmation of MTC. In these cases, haplotype analysis excluded the involvement of the RET gene itself in the pathogenesis of the MTC. In three patients, the coexistence, in different foci, of medullary and papillary thyroid cancer was documented. The genetic studies did not show ret/PTC rearrangements. The microsatellite analysis excluded co‐segregation of RET locus with the MTC/PTC phenotype.

conclusions We report a full clinical and molecular analysis of a large FMTC kindred with an uncommon RET mutation. In two family members, phenotype and genotype were not concordant, representing the first evidence of FMTC phenocopies. Furthermore, the association of familial forms of medullary and papillary thyroid cancers has been found in 30% of patients undergoing thyroidectomy for MTC. In these situations, genetic analyses excluded the possible germline involvement of RET. Though FMTC phenocopies are likely to represent an exceptional finding, such a possibility should be taken into account in the genetic counselling for MEN 2 syndromes.
Study of diurnal fluctuations of plasma methoxyamines in healthy volunteers
Authors: Hoizey Guillaume , Lukas‐Croisier Céline , Frances Carole , Grulet Hervé , Delemer Brigitte , Millart Hervé , Devillier Philippe , Caron Jean
Published: March 2003

objective We studied the diurnal fluctuations of plasma concentrations of methoxyamines (metanephrine and normetanephrine) and of their parent amines (epinephrine and norepinephrine) in normotensive subjects.

design Serial blood sampling at 09·00 h, 11·00 h, 12·00 h, 14·00 h, 16·00 h, 18·00 h and 20·00 h in 28 healthy volunteers at rest. Determination of plasma concentrations of free catecholamines and total methoxyamines (free and sulpho‐conjugates) was carried out by high‐performance liquid chromatography with electrochemical detection.

results Mean (± SD) plasma concentrations of total metanephrine (MN) and normetanephrine (NMN) were 4·31 ± 1·73 nmol/l (range: 0·96–9·3 nmol/l) and 8·13 ± 2·54 nmol/l (range: 3·14–17·0 nmol/l), respectively. The NMN/MN ratio ranged between 0·8 and 7·8 (mean ± SD 2·1 ± 1·0). Mean plasma concentrations of free epinephrine and norepinephrine were 0·21 ± 0·12 nmol/l (range: 0·06–1·39 nmol/l) and 1·61 ± 0·62 nmol/l (range: 0·47–4·01 nmol/l), respectively. Despite marked intraindividual fluctuations, mean methoxyamine and catecholamine levels remained constant over the entire duration of the experiment.

conclusions The absence of fluctuations of plasma levels of total methoxyamines suggests that their measurement could be carried out at any time within the diurnal time frame. Further investigations, however, remain necessary to validate these findings in patients with hypertension and/or pheochromocytoma, and to explain the ever important intraindividual variation in plasma concentrations of methoxyamines and of their parent compounds.
Learning from previous responses in phase I dose‐escalation studies
Authors: Whitehead John , Zhou Yinghui , Stallard Nigel , Todd Susan , Whitehead Anne
Published: March 2003

Dose escalation in phase I studies is generally performed on the basis of clinical experience and judgement. In this paper some of the statistical approaches that have been proposed for the formalization of the procedure are described. Apart from the use of the Continual Reassessment Method in oncology studies, such formal methods have received little implementation. The purpose of presenting them here is to promote their further exploration and appropriate implementation. Certain limitations are discussed, which will be best overcome by collaboration between clinical pharmacologists and statisticians.
A 6‐month large‐scale study into the safety of tamsulosin
Authors: Michel M. C. , Bressel H.‐U. , Goepel M. , Rübben H.
Published: March 2003

Aims  Tamsulosin is an α1‐adrenoceptor antagonist for the treatment of symptomatic benign prostatic hyperplasia with a tolerability similar to that of placebo in short‐term, placebo‐controlled studies with limited patient numbers. The present study was designed to test the safety of tamsulosin treatment in a large cohort of men during a prolonged period of time, particularly with regard to comedications.

Methods  A multicentre, open‐label phase IIIb study with 1784 patients receiving 0.4 mg o.d. tamsulosin for 6 months was performed according to good clinical practice guidelines. The analysis was performed on an intention‐to‐treat basis and powered to detect adverse events (AE) occurring in 0.15% of patients with 95% confidence.

Results  During a total drug exposure time of 811 patient years, 386 AE were recorded in 253 patients (14.2%; 95% confidence intervals [CI] 12.0–15.2%). Twenty‐nine patients suffered 44 serious AE including five fatal events (CI 0.12–0.73%) due to myocardial infarction (n = 3) and to pneumonia and a car accident (one each), but all deaths were judged to be unlikely to be related to study medication. The frequency of AE in patients without any comedication (n = 1095) was 13.0% (CI 11.3–14.9%). In a logistic regression analysis β‐adrenoceptor blockers, converting enzyme inhibitors, antidiabetics and diuretics did not significantly affect the odds ratio for having AE. However, concomitant α‐adrenoceptor antagonists (a protocol violation) and treatment with verapamil (which also has α‐adrenoceptor antagonist activity) significantly enhanced the odds ratio for having AE to 3.87 (CI 1.52–9.85) and 3.17 (CI 1.52–6.58), respectively. Minor increases in the odds ratio, which did not reach statistical significance, were also observed for Ca2+ antagonists other than verapamil and for nitrates.

Conclusions  We conclude that tamsulosin has a good safety profile relative to AE rates in the placebo arms of previous studies on tamsulosin even in the presence of most potentially complicating comedications. No major unexpected severe AE were recorded during our 6 months study.
Comparison of the effects of nadolol and bisoprolol on the isoprenaline‐evoked dilatation of the dorsal hand vein in man
Authors: Abdelmawla A. H. , Langley R. W. , Szabadi E. , Bradshaw C. M.
Published: March 2003

Aims  We attempted to explore the possible differential involvement of β‐adrenoceptor subtypes in the dilator response of the human dorsal hand vein to isoprenaline by examining the ability of bisoprolol, a selective β1‐adrenoceptor antagonist, and nadolol, a nonselective β1/β2‐adrenoceptor antagonist, to antagonize the response.

Methods  Twelve healthy male volunteers participated in four weekly sessions. In the preliminary session a dose‐response curve to the vasoconstrictor effect of phenylephrine was constructed and the dose producing 50–75% maximal response was determined for each individual. In each of the remaining three (treatment) sessions, nadolol (40 mg), bisoprolol (5 mg) or placebo was ingested, and isoprenaline hydrochloride (3.33–1000 ng min−1) was infused locally into the dorsal hand vein along with a constant dose of phenylephrine hydrochloride (to preconstrict the vein) 2 h after the ingestion of the drugs. Changes in vein diameter were monitored with the dorsal hand vein compliance technique. Subjects were allocated to treatment session according to a double‐blind balanced cross‐over design. Systolic and diastolic blood pressure, and heart rate were also measured.

Results  Isoprenaline produced dose‐dependent venodilatation which was antagonized by nadolol but remained unaffected by bisoprolol (anova with repeated measures: P < 0.025; Dunnett's test: placebo vs nadolol, P < 0.01; placebo vs bisoprolol, P = NS). Mean log ED50 (ng min−1) was significantly increased in the presence of nadolol and remained unchanged in the presence of bisoprolol (anova, P < 0.025; Dunnett's test: placebo vs nadolol, P < 0.005; placebo vs bisoprolol, P = NS; differences between mean log ED50[95% CI]: placebo vs bisoprolol −0.11 [‐0.38, 0.16], placebo vs nadolol 0.32[0.09, 0.72], bisoprolol vs nadolol −0.43 [‐0.71, −0.15]). Mean Emax did not differ in the three treatment conditions.

Conclusions

 The failure of bisoprolol to attenuate isoprenaline‐evoked venodilatation in the human dorsal hand vein argues against the involvement of a β1‐adrenoceptor‐mediated component in the isoprenaline‐evoked venodilatory responses. The possibility cannot be excluded that the consequences of β1‐adrenoceptor blockade by bisoprolol might have been obscured by a possible venodilator effect of bisoprolol.
In vitro metabolism and drug interaction potential of a new highly potent anti‐cytomegalovirus molecule, CMV423 (2‐chloro 3‐pyridine 3‐yl 5,6,7,8‐tetrahydroindolizine 1‐carboxamide)
Authors: Bournique Bruno , Lambert Nicole , Boukaiba Rachid , Martinet Michel
Published: March 2003

Aims
 To identify the enzymes involved in the metabolism of CMV423, a new anticytomegalovirus molecule, to evaluate its in vitro clearance and to investigate its potential involvement in drug/drug interactions that might occur in the clinic.

Methods
 The enzymes involved in and the kinetics of CMV423 biotransformation were determined using pools of human liver subcellular fractions and heterologously expressed human cytochromes P450 (CYP) and FMO. The effect of CMV423 on CYP probe activities as well as on indinavir and AZT metabolism was determined, and 26 drugs were tested for their potential to inhibit or activate CMV423 metabolism.

Results
 CMV423 was oxidized by CYP and not by FMO or cytosolic enzymes. The Km values for 8‐hydroxylation to rac‐RPR 127025, an active metabolite, and subsequent ketone formation by human liver microsomes were 44 ± 13 µm and 47 ± 11 µm, respectively, with corresponding Vmax/Km ratios of 14 and 4 µl min−1 nmol−1 P450. Inhibition with selective CYP inhibitors indicated that CYP1A2 was the main isoform involved, with some participation from CYP3A. Expressed human CYP1A1, 1A2, 2C9, 3A4 and 2C8 catalysed rac‐RPR 127025 formation with Km values of < 10 µm, 50 ± 21 µm, 55 ± 19 µm, circa 282 ± 61 µm and circa 1450 µm, respectively. CYP1B1, 2A6, 2B6, 2C19, 2D6, 2E1 or 3A5 did not catalyse the reaction to any detectable extent. CYP1A1 and 3A4 also catalysed ketone formation from rac‐RPR 127025. In human liver microsomes, CMV423 at 1 and 10 µm inhibited CYP1A2 activity up to 31% and 63%, respectively, CYP3A4 activity up to 40% (10 µm) and CYP2C9 activity by 35% (1 and 10 µm). No effect was observed on CYP2A6, 2D6 and 2E1 activities. CMV423 had no effect on indinavir and AZT metabolism. Amongst 26 drugs tested, none inhibited CMV423 metabolism in vitro at therapeutic concentrations.

Conclusions
 CMV423 is mainly metabolized by CYP1A2 and 3A4. Its metabolism should not be saturable at the targeted therapeutic concentrations range (Cmax < 1 µm). CMV423 will probably affect CYP1A2 and 1A1 activities in vivo to some extent, but no other drug–drug interactions are expected.
The effects of an oral contraceptive containing ethinyloestradiol and norgestrel on CYP3A activity
Authors: Belle Donna J. , Callaghan John T. , Gorski J. Christopher , Maya Juan F. , Mousa Omiema , Wrighton Steven A. , Hall Stephen D.
Published: March 2003

Aims  To examine the effects of an oral contraceptive containing ethinyloestradiol and norgestrel on intestinal and hepatic CYP3A activity using midazolam as a probe substrate.

Methods  In a nonblinded sequential study, nine healthy women received simultaneous doses of intravenous midazolam (0.05 mg kg−1) and oral 15N3‐midazolam (3 mg) on days 0, 4, 6, 8, and 14. On study day 5, Ovral™ (50 µg ethinyloestradiol/500 µg norgestrel) was administered for 10 days. Serum and urine samples were assayed for midazolam, 15N3‐midazolam and metabolites by liquid chromatography‐mass spectrometry. A Digit Symbol Substitution Test (DSST) was used to assess changes in the pharmacodynamic activity of midazolam.

Results  Moderate (% CV 26–46) interindividual variability in the pharmacokinetics of midazolam were observed. Compared with baseline, AUC(0,∞)iv ratios (95% CIs) after 2, 4, and 10 days treatment with OC were 89% (79, 101), 96% (85, 109), and 88% (77, 99), respectively. The AUC(0,∞)oral ratios (95% CIs) were 101% (82, 125), 105% (85, 130), and 114% (92, 141), respectively, after 2, 4, and 10 days OC treatment compared with baseline. Concomitant administration of the oral contraceptive, Ovral™ for 2, 4 or 10 days did not significantly alter the area under the curve, clearance, or half‐life of midazolam after either oral or intravenous administration. No alterations in pharmacodynamic effects of midazolam were observed between treatment days. Mean DSST scores strongly correlated with mean total midazolam blood concentrations (r = −0.936).

Conclusions  Administration of Ovral™ for 10 days had no impact on intestinal or hepatic CYP3A activity as determined by midazolam metabolism.
Maximizing the value of medicines by including pharmacogenetic research in drug development and surveillance
Authors: Brazell C. , Freeman A. , Mosteller M.
Published: March 2003

Genetics provides significant opportunities to maximize the safety and efficacy of medicines. Over the next 3–5 years, it may be possible to develop tools that use selective information from patients' DNA to enable healthcare professionals to predict more accurately those patients at risk of serious adverse events to some medicines currently available. This is likely to be followed, over the next 5–10 years, by the application of the technology to predict more accurately if individual patients will obtain a therapeutic benefit from a particular medicine. The ability to accurately predict patient response will inevitably change the way medicines are developed, evaluated, and prescribed. Advances in single nucleotide polymorphism (SNP) map technology are likely to drive this innovation. Abbreviated SNP profiles will provide the means to define medicine response tests, thereby allowing clinicians to select the medicine to which the patient is likely to gain the greatest benefit and least risk. This will help to maximize efficacy and reduce the incidence of drug‐related adverse events. It may be possible to identify SNP profiles during larger Phase II clinical trials which predict efficacy, and use these to form the basis of Phase III entry criteria. As a result, Phase III trials may be streamlined for many medicines making them smaller, more efficient, and more focused. In addition it may be possible to incorporate pharmacogenetics into postmarketing surveillance strategies to provide a means to identify SNPs which predict uncommon serious adverse drug reactions, and so refine the initial medicine response test. The ability to develop drugs with a predictable response will allow clinicians to provide targeted treatment for patients, with greater confidence of safety and efficacy. Patients therefore will receive more efficacious, timely, and well‐tolerated medicines. The challenge for those involved in drug development is to model and evaluate the application of pharmacogenetics so that steps can be taken to realize this potential.
Quaternary alluvial fan sedimentation in the Dehradun Valley Piggyback Basin, NW Himalaya: tectonic and palaeoclimatic implications
Authors: Kumar Singh A. , Parkash B. , Mohindra R. , Thomas J. V. , Singhvi A. K.
Published: March 2003

ABSTRACT The Dehradun Valley, a synclinal intermontane valley piggyback basin within the Siwalik Group rocks in the NW Himalaya, is separated from the Lesser Himalayan formations in the north by a major intraplate thrust, the Main Boundary Thrust (MBT) and from the Indogangetic Plains in the south by the Himalayan Frontal Fault (HFF). Major parts of the Dehradun Valley are covered by three fans, from west to east the Donga, Dehradun and Bhogpur fans, deposited by streams following the topography produced by activity of the MBT and probable footwall imbricate thrusts, starting at about 50 ka.

The Donga and Dehradun fans were fed by small streams and characterized mainly by sediment gravity‐flow deposits (debris flow and mudflow deposits) in the proximal zone, and mostly mudflow deposits and minor braided stream deposits in the middle zone during the period 50–10 ka. Palaeosols were weakly developed in the proximal zone and moderately to strongly developed in the middle zone. The degree of development of palaeosol was mainly a function of rate of sedimentation and to some extent entrenchment of streams into the fan surface. Since 10 ka, deposition has been typically by braided streams. The Bhogpur fan has been marked by deposition from relatively larger braided streams since 50 ka.

The fan sequences in the Dehradun Valley are synorogenic and their deposition started due to activity of the southern footwall imbricate of the MBT, i.e. Bhauwala Thrust on the Donga and Dehradun fans. In these fans, major fan sequences show retrogradation (50–10 ka) related to a decrease in the activity of the MBT and related imbricates and activity of more hinterlandward imbricates with time. After 10 ka a thin prograding sequence was deposited due to uplift of the fans, which resulted from the activity on a thrust in the distal parts of the fans. It suggests an out‐of‐sequence activity of faults in the MBT imbricate system. Cross‐faults divide the Siwalik formations in the footwall of the MBT into three blocks, which were marked by decreasing subsidence or possibly uplift from east to west. Thrusting on the HFF was not piggyback type but synchronous with activity of the MBT and its imbricates. The development of the Mohand fault‐bend anticline above the HFF changed the nature of the basin from foreland to piggyback type, shed minor colluvial deposits prior to 10 ka, and folded the southernmost fan deposits in the western, narrow parts of the valley.

A major change in climate from a cold, dry climate with strong seasonal variations prevailing since 50 ka to warm and humid climate at about 10 ka resulted in a change in depositional processes from sediment gravity‐flows to braided streams.