bluuebunny/crossref_metadata_2025 huggingface dataset:
A new siglec family member, siglec‐10, is expressed in cells of the immune system and has signaling properties similar to CD33
Authors: Whitney Gena , Wang Shulin , Chang Han , Cheng Ke‐Yi , Lu Pin , Zhou Xia D. , Yang Wen‐Pin , McKinnon Murray , Longphre Malinda
Published: March 2003

The siglecs (sialic acid‐binding Ig‐like lectins) are a distinct subset of the Ig superfamily with adhesion‐molecule‐like structure. We describe here a novel member of the siglec protein family that shares a similar structure including five Ig‐like domains, a transmembrane domain, and a cytoplasmic tail containing two ITIM‐signaling motifs. Siglec‐10 was identified through database mining of an asthmatic eosinophil EST library. Using the Stanford G3 radiation hybrid panel we were able to localize the genomic sequence of siglec‐10 within the cluster of genes on chromosome 19q13.3‐4 that encode other siglec family members. We have demonstrated that siglec‐10 is an immune system‐restricted membrane‐bound protein that is highly expressed in peripheral blood leukocytes as demonstrated by Northern, RT‐PCR and flow cytometry. Binding assays determined that the extracellular domain of siglec‐10 was capable of binding to peripheral blood leukocytes. The cytoplasmic tail of siglec‐10 contains four tyrosines, two of which are embedded in ITIM‐signaling motifs (Y597 and Y667) and are likely involved in intracellular signaling. The ability of tyrosine kinases to phosphorylate the cytoplasmic tyrosines was evaluated by kinase assay using wild‐type siglec‐10 cytoplasmic domain and Y→F mutants. The majority of the phosphorylation could be attributed to Y597 andY667. Further experiments with cell extracts suggest that SHP‐1 interacts with Y667 and SHP‐2 interacts with Y667 in addition to another tyrosine. This is very similar to CD33, which also binds the phosphatases SHP‐1 and SHP‐2, therefore siglec‐10, as CD33, may be characterized as an inhibitory receptor.
The interferon alpha induced protein ISG12 is localized to the nuclear membrane
Authors: Martensen Pia M. , Søgaard T. Max M. , Gjermandsen Irene M. , Buttenschøn Henriette N. , Rossing Anette B. , Bonnevie‐Nielsen Vagn , Rosada Cecilia , Simonsen Janne L. , Justesen Just
Published: March 2003

Interferons exert their biological function mainly through the activation of interferon‐stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon α inducible genes of unknown function. We have determined the 5′ end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon α inducibility of the gene. Subsequently, we have expressed human ISG12, a 12‐kDa hydrophobic protein in the baculovirus expression system and with a C‐terminal FLAG‐tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon α by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope.
ATP causes small heat shock proteins to release denatured protein
Authors: Wang Keyang , Spector Abraham
Published: March 2003

Small heat‐shock proteins (sHSPs) are a ubiquitous family of low molecular mass (15–30 kDa) stress proteins that have been found in all organisms. Under stress, sHSPs such as α‐crystallin can act as chaperones binding partially denatured proteins and preventing further denaturation and aggregation. Recently, it has been proposed that the function of sHSPs is to stabilize stress‐denatured protein and then act cooperatively with other HSPs to renature the partially denatured protein in an ATP‐dependent manner. However, the process by which this occurs is obscure. As no significant phosphorylation of α‐crystallin was observed during the renaturation, the role of ATP is not clear. It is now shown that ATP at normal physiological concentrations causes sHSPs to change their confirmation and release denatured protein, allowing other molecular chaperones such as HSP70 to renature the protein and renew its biological activity. In the absence of ATP, sHSPs such as α‐crystallin are more efficient than HSP70 in preventing stress‐induced protein aggregation. This work also indicates that in mammalian systems at normal cellular ATP concentrations, sHSPs are not effective chaperones.
Subcellular distribution of 14‐3‐3 proteins in the unicellular green alga Chlamydomonas reinhardtii
Authors: Voigt Jürgen , Liebich Ines , Kieß Michael , Frank Ronald
Published: March 2003

A polyclonal antibody was raised against a recombinant Chlamydomonas 14‐3‐3–β‐galactosidase (β‐Gal) fusion protein and characterized for its epitope specificity towards the corresponding Chlamydomonas 14‐3‐3 protein by scan‐peptide analysis. This antibody recognized four Chlamydomonas polypeptides with apparent molecular masses 32, 30, 27, and 24 kDa, which also reacted with the antiserum depleted of anti‐(Escherichia coliβ‐Gal) IgG, but not with the corresponding preimmune serum or the antiserum preincubated with purified 14‐3‐3 proteins. Western‐blot analyses performed with the antibody depleted of anti‐(β‐Gal) IgG revealed that more or less pronounced levels of 14‐3‐3 proteins were present in all subcellular fractions of Chlamydomonas reinhardtii except the nuclei. The highest levels of 14‐3‐3 protein were observed in the cytosol and microsomal fraction. The 30‐kDa isoform was predominant in the cytosol, whereas the 27‐kDa isoform was prevalent in the microsomes. When microsomal membranes were separated by sucrose‐density‐gradient centrifugation, Western‐blot analysis revealed distinct patterns of 14‐3‐3 isoforms in the endoplasmic reticulum, dictyosome, and plasma membrane fractions identified by marker enzyme activities. These findings indicate that the four 14‐3‐3 proteins of C. reinhardtii differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane.
Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain
Authors: Geyer Hildegard , Bahr Ute , Liedtke Steffen , Schachner Melitta , Geyer Rudolf
Published: March 2003

Polysialylation of the neural cell adhesion molecule (N‐CAM) is known to destabilize cell‐cell adhesion and to promote plasticity in cell–cell interactions. To gain more insights into the molecular mechanisms regulating the selective expression of polysialic acid on distinct glycan chains, the underlying core structures of polysialylated N‐CAM glycans from newborn mouse brain were examined. Starting from low picomolar amounts of oligosaccharides, a multistep approach was used that was based on various mass spectrometric techniques with minimized sample consumption. Evidence could be provided that polysialylated murine N‐CAM glycans comprise diantennary, triantennary and tetraantennary core structures carrying, in part, type‐1 N‐acetyllactosamine antennae, sulfate groups linked to terminal galactose or subterminal N‐acetylglucosamine residues and, as a characteristic feature, a sulfated glucuronic acid unit which was bound exclusively to C3 of terminal galactose in Manα3‐linked type‐2 antennae. Hence, our results reveal that part of the murine N‐CAM carbohydrates are modified within a single oligosaccharide by polysialic acid plus a HSO3‐GlcA‐moiety, which is likely to represent a HNK1‐epitope. As HNK1‐carbohydrates are also known to modulate cell–cell interactions, the simultaneous presence of both carbohydrate epitopes may reflect a new mechanism involved in the fine‐tuning of N‐CAM functions.
Structure–function analysis of CYP27B1 and CYP27A1
Authors: Sawada Natsumi , Sakaki Toshiyuki , Kitanaka Sachiko , Kato Shigeaki , Inouye Kuniyo
Published: March 2003

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D‐dependent rickets type I (VDDR‐I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653–661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111–4117]. None of the CYP27B1 mutants showed 1α‐hydroxylase activity towards 25‐hydroxyvitamin D3. Thus, it was assumed that the mutated amino‐acid residues play important roles in the 1α‐hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme‐binding and substrate‐binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO‐difference spectra and substrate‐induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure–function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino‐acid residues destroyed the tertiary structure of the substrate‐heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme–propionate binding, and Asp164 stabilized the four‐helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.
Cloning and expression of sterol Δ14‐reductase from bovine liver
Authors: Roberti Rita , Bennati Anna Maria , Galli Giovanni , Caruso Donatella , Maras Bruno , Aisa Cristina , Beccari Tommaso , Della Fazia Maria Agnese , Servillo Giuseppe
Published: March 2003

Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes. Sterol Δ14‐reductase (Δ14‐SR) is a microsomal enzyme involved in the conversion of lanosterol to cholesterol in mammals. Amino‐acid sequence analysis of a 38‐kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR‐1, encoded by the TM7SF2 gene, and with the C‐terminal domain of human lamin B receptor. A cDNA encoding the 38‐kDa protein, similar to human TM7SF2, was identified by analysis of␣a␣bovine expressed sequence tag (EST) database. The␣cDNA was synthesized by RT‐PCR, cloned, and sequenced. The cDNA encodes a 418 amino‐acid polypeptide with nine predicted transmembrane domains. The deduced amino‐acid sequence exhibits high similarity with Δ14‐SR from yeasts, fungi, and plants (55–59%), suggesting that the bovine cDNA encodes Δ14‐SR. Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain. The polypeptide encoded by the cloned cDNA was expressed in COS‐7 cells. Immunofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS‐7 cells expressing the protein exhibited Δ14‐SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Δ14‐SR and provide evidence that the human TM7SF2 gene encodes Δ14‐SR.
Early growth response‐1 gene (Egr‐1 ) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro
Authors: Meyer Ralph G. , Küpper Jan‐Heiner , Kandolf Reinhard , Rodemann H. Peter
Published: March 2003

The promoter of the early growth response gene (Egr‐1) has been described to be activated by ionizing radiation, and it seems to be clear that this process involves different mitogen activated protein (MAP) kinases, dependent on the specific cell type examined. However, early steps leading to activation of the corresponding pathways and thus to overexpression of Egr‐1 are not well understood. In this study, deletion mutants of the 5′ upstream region of the Egr‐1 gene were generated which allowed us to correlate the radiation–induction of the Egr‐1 promoter in U87 glioma cells to five serum response elements. Based␣on the data shown, a possible role of two cAMP responsive elements for radiation‐dependent promoter regulation could be ruled out. On the basis of activator/inhibitor studies applying fetal bovine serum, EGF, PD98059, anisomycin, SB203580, forskolin and wortmannin, it could be demonstrated that in U87 cells the ERK1/2 and␣potentially SAPK/JNK, but not the p38MAPK/SAPK2, pathway contribute to the radiation‐induction of Egr‐1 promoter. In addition, it was observed that irradiated cells secrete a diffusible factor into the culture media␣which accounts for the radiation‐induced promoter upregulation. By blocking growth factor receptor activation with suramin, this effect could be completely abolished.
Purification and properties of an alkaline proteinase of Fusarium culmorum
Authors: Pekkarinen Anja I. , Jones Berne L. , Niku‐Paavola Marja‐Leena
Published: March 2003

The disease Fusarium head blight (scab) causes severe problems for farmers and for the industries that use cereals. It is likely that the fungi that cause scab (Fusarium spp.) use various enzymes when they invade grains. We are studying enzymes that the fungi may use to hydrolyze grain proteins. To do this, Fusarium culmorum was grown in a gluten‐containing medium from which an alkaline serine proteinase with a molecular mass of 28.7 kDa was purified by size‐exclusion and cation exchange chromatographies. The enzyme was maximally active at pH 8.3–9.6 and 50 °C, but was unstable under these conditions. It hydrolyzed the synthetic substrates N‐succinyl‐Ala‐Ala‐Pro‐Phe p‐nitroanilide and, to a lesser extent, N‐succinyl‐Ala‐Ala‐Pro‐Leu p‐nitroanilide. It was inhibited by phenylmethanesulfonyl fluoride and chymostatin, but not by soybean trypsin or Bowman–Birk inhibitors. Parts of the amino‐acid sequence were up to 82% homologous with those of several fungal subtilisins. One of the active site amino acids was detected and it occupied the same relative position as in the other subtilisins. Therefore, on the basis of these characteristics, the proteinase is subtilisin‐like. Purification of the enzyme was complicated by the fact that, when purified, it apparently underwent autolysis. The presence of extraneous protein stabilized the activity.
Exploring the primary electron acceptor (QA)‐site of the bacterial reaction center from Rhodobacter sphaeroides
Authors: Hucke Oliver , Schmid Ralf , Labahn Andreas
Published: March 2003

The functional replacement of the primary ubiquinone (QA) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides with synthetic vitamin K derivatives has provided a powerful tool to investigate the electron transfer mechanism. To investigate the binding mode of these quinones to the QA binding site we have determined the binding free energy and charge recombination rate from QA– to D+ (kAD) of 29 different 1,4‐naphthoquinone derivatives with systematically altered structures. The most striking result was that none of the eight tested compounds carrying methyl groups in both positions 5 and 8 of the aromatic ring exhibited functional binding. To understand the binding properties of these quinones on a molecular level, the structures of the reaction center‐naphthoquinone complexes were predicted with ligand docking calculations. All protein–ligand structures show hydrogen bonds between the carbonyl oxygens of the quinone and AlaM260 and HisM219 as found for the native ubiquinone‐10 in the X‐ray structure. The center‐to‐center distance between the naphthoquinones at QA and the native ubiquinone‐10 at QB (the secondary electron acceptor) is essentially the same, compared to the native structure. A detailed analysis of the docking calculations reveals that 5,8‐disubstitution prohibits binding due to steric clashes of the 5‐methyl group with the backbone atoms of AlaM260 and AlaM249. The experimentally determined binding free energies were reproduced with an rmsd of ≈ 4 kJ·mol−1 in most cases providing a valuable tool for the design of new artificial electron acceptors and inhibitors.
Myristyl and palmityl acylation of pI 5.1 carboxylesterase from porcine intestine and liver
Authors: Smialowski‐Fléter Sylvie , Moulin André , Perrier Josette , Puigserver Antoine
Published: March 2003

Immunoblotting analyses revealed the presence of carboxylesterase in the porcine small intestine, liver, submaxillary and parotid glands, kidney cortex, lungs and cerebral cortex. In the intestinal mucosa, the pI 5.1 enzyme was detected in several subcellular fractions including the microvillar fraction. Both fatty monoacylated and diacylated monomeric (F1), trimeric (F3) and tetrameric (F4) forms of the intestinal protein were purified here for the first time by performing hydrophobic chromatography and gel filtration. The molecular mass of these three enzymatic forms was␣estimated to be 60, 180 and 240 kDa, respectively, based on size‐exclusion chromatography and SDS/PAGE analysis. The existence of a covalent attachment linking palmitate and myristate to porcine intestinal carboxylesterase (PICE), which was suggested by the results of gas‐liquid chromatography (GLC) experiments in which the fatty acids resulting from alkali treatment of the protein forms were isolated, was confirmed here by the fact that [3H]palmitic and [3H]myristic acids were incorporated into porcine enterocytes and hepatocytes in cell primary cultures. Besides these two main fatty acids, the presence of oleic, stearic, and arachidonic acids was also detected by GLC and further confirmed by performing radioactivity counts on the 3H‐labelled PICE forms after an immunoprecipitation procedure using specific polyclonal antibodies, followed by a SDS/PAGE separation step. Unlike the F1 and F4 forms, which were both myristoylated and palmitoylated, the F3 form was only palmitoylated. The monomeric, trimeric and tetrameric forms of PICE were all able to hydrolyse short chain fatty acids containing glycerides, as well as phorbol esters. The broad specificity of fatty acylated carboxylesterase is discussed in terms of its possible involvement in the metabolism of ester‐containing xenobiotics and signal transduction.
Mitogen‐activated protein kinase kinase negatively modulates ciliary neurotrophic factor‐activated choline acetyltransferase gene expression
Authors: Mellott Tiffany , Lopez‐Coviella Ignacio , Blusztajn Jan Krzysztof , Berse Brygida
Published: March 2003

The expression of the choline acetyltransferase (ChAT) enzyme that synthesizes the neurotransmitter acetylcholine (ACh) is upregulated by ciliary neurotrophic factor (CNTF). We studied the involvement of the mitogen‐activated protein kinase (MAPK) pathway in regulating ChAT expression in a murine septal cell line. Surprisingly, we found that PD98059 and U0126, two structurally distinct inhibitors of MAPK kinase (MEK1), increased both basal and CNTF‐induced ACh production. Transient transfections with ChAT promoter‐luciferase reporter construct demonstrated synergy between PD98059 and CNTF at the transcriptional level. Moreover, in cotransfection studies, overexpression of constitutively activated MEK1 completely abrogated the CNTF‐mediated induction of the reporter. Blocking MEK1 did not significantly alter CNTF‐induced Tyr705 phosphorylation of the principal mediator of the CNTF pathway, the transcription factor Stat3. However, PD98059 inhibited Ser727 phosphorylation of Stat3, demonstrating that the latter is MEK1‐dependent. Taken together, these results indicate that activation of the MEK1/MAPK pathway inhibits the CNTF‐mediated stimulation of ChAT expression, possibly as a part of a feedback mechanism.
Leaf dark respiration as a function of canopy position in Nothofagus fusca trees grown at ambient and elevated CO2 partial pressures for 5 years
Authors: Griffin K. L. , Tissue D. T. , Turnbull M. H. , Schuster W. , Whitehead D.
Published: March 2003

Mass‐based and area‐based rates of respiration, leaf nitrogen content, leaf total protein content, non‐structural carbohydrates and leaf mass per unit area (LMA) all decreased with depth in the canopy of Nothofagus fusca (Hook. F.) Oerst. (Red beech) trees grown for 5 years at ambient (36 Pa) or elevated (66 Pa) CO2 partial pressures.

Elevated CO2 partial pressure had a strong effect on dark respiration, decreasing both mass‐based and area‐based rates at all canopy positions, but had little or no effect on leaf physical and biochemical properties.

Leaf sugars, starch, protein, N and LMA were all correlated with respiration rate, and are therefore strong predictors of area‐based dark respiration rates. The y axis intercept of regressions of respiration rate on mean leaf N, protein, starch and LMA was lower for plants grown at elevated compared to ambient CO2 partial pressures because of the differential effect of growth at elevated CO2 partial pressure on leaf gas‐exchange, chemical and physical characteristics.

 The lower respiration rates for leaves from trees grown at elevated CO2 partial pressure resulted in a significant increase in the ratio of light‐saturated net photosynthesis to respiration, increasing the potential carbon‐use efficiency of these leaves.
Interspecific variation in potential water‐use efficiency and its relation to plant species abundance in a water‐limited grassland
Authors: Tsialtas J. T. , Handley L. L. , Kassioumi M. T. , Veresoglou D. S. , Gagianas A. A.
Published: March 2003

Interspecific and temporal variations in presumed water‐use efficiency, as measured by carbon isotope discrimination (Δ), were examined in a Greek semiarid herbaceous grassland, and the hypothesis tested that competition for water is a factor in determining vegetation structure.

 Experiments were conducted for 3 years in an upland grassland consisting of exclusively C3perennial species. The most abundant species were the grassesPoa pratensisL.,Lolium perenneL. andFestuca valida(Uechtr.) Penzes; the forbsAchillea millefoliumL.,Plantago lanceolataL. andTaraxacum officinaleWeber ex Wigg; and the legumeTrifolium repensL.

 Rooting depth did not explain plant Δ in this shallow‐soil site, where soil moisture was concentrated in the top of the profile.

 Although temporal variation for Δ was significant within most species, changes in ranking of species were slight throughout three growing seasons.

 Δ Was negatively related to species biomass production, which means that species’ abundances were positively related to species’ water‐use efficiencies. Δ Was positively related to leaf N content, which we interpret as an autocorrelation with water availability.

 The results of this study are consistent with the notion that, in an environment where water is scarce, plants may compete effectively by increasing their potential water‐use efficiency as measured by Δ, and that species abundance is regulated by competition‐driven water depletion.
A self‐assembling model of resource dynamics and plant growth incorporating plant functional types
Authors: Colasanti R. L. , Hunt R. , Askew A. P.
Published: March 2003

We explored the ecological properties of a self‐assembling, cellular automaton model by simulating the behaviour of distinct plant functional types.

 Types differed according to three fundamental traits: size of plant modules; longevity of modules in the absence of resources; and propensity to flowering.

 When expressed at three levels apiece, the three traits were able to approximate the essential biology of seven important types within a well known system of plant functional types (the C‐S‐R system, based on disturbance and resource availability).

 The seven types, when grown in real experiments with virtual communities, reproduced classical community‐level behaviour to the extent of demonstrating smooth, competitive replacements along gradients of resource or disturbance, or both.

 In complex communities simulated over a wide range of environmental conditions, the seven types produced a clearly ‘humpbacked’ curve linking plant diversity to community productivity. The mixtures of species resulting from these experiments together represented all parts of C‐S‐R space.

 These outcomes emerged solely from the ‘bottom‐up’ processes inherent in self‐assembling cellular automata. No community‐level specifications were inserted into the model, yet the community‐level consequences agreed with both the underlying predictions of C‐S‐R theory and field observations.

 This work confirms that the dynamics of herbaceous plant communities can be emergent from the resource‐handling properties of the organs of the constituent species, and that a simple rule base can, to a first approximation, be sufficient to reproduce such dynamics.
Effects of low‐dose oral hydrocortisone replacement versus short‐term reproduction of physiological serum cortisol concentrations on insulin action in adult‐onset hypopituitarism
Authors: McConnell E. M. , Bell P. M. , Ennis C. , Hadden D. R. , McCance D. R. , Sheridan B. , Atkinson A. B.
Published: March 2003

objective Hypercortisolism is associated with impaired glucose tolerance and insulin resistance. For many years hydrocortisone 30 mg was the standard total daily replacement dose in adult hypopituitarism. The use of this conventional dose has now been shown to result in mild biochemical hypercortisolism and might contribute to the increased cardiovascular risk reported in hypopituitarism. The use of lower doses of hydrocortisone replacement therapy might prevent some of the adverse metabolic effects seen with conventional doses.

patients In a randomized crossover study we assessed peripheral and hepatic insulin action in 15 ACTH‐deficient patients with normal glucose tolerance on two occasions while receiving either a low‐dose oral hydrocortisone replacement (LOR) therapy (15 mg at 0800, 5 mg at 1700) or a physiological hydrocortisone infusion (PHI), which achieved physiological serum cortisol concentrations.

results Exogenous glucose infusion rates required to maintain euglycaemia were similar for the LOR and the PHI protocols (26·2 ± 0·4 vs. 23·8 ± 0·6 µmol/kg/min, respectively). Endogenous glucose production was also similar (12·0 ± 2·5 vs. 11·6 ± 2·4 µmol/kg/min, respectively) and in the post‐absorptive state suppressed to a similar extent following insulin (4·5 ± 2·0 vs. 5·1 ± 3·1 µmol/kg/min).

conclusion Hydrocortisone replacement therapy at a dose of 15 mg with breakfast, 5 mg with evening meal does not increase peripheral or hepatic insulin resistance when compared to a hydrocortisone infusion designed to simulate physiological serum cortisol concentrations.
Correlation between midazolam and lignocaine pharmacokinetics and MEGX formation in healthy volunteers
Authors: Swart Eleonora L. , van der Hoven Ben , Johan Groeneveld A. B. , Touw Daniel J. , Danhof Meindert
Published: March 2003

Aims  The objectives of the present investigation were: (a) to determine the correlation between lignocaine and midazolam pharmacokinetics following intravenous administration in healthy volunteers, (b) to determine the effects of treatment with an inhibitor of CYP3A4 (erythromycin) on this correlation and (c) to assess the precision of the MEGX‐test as a sole predictor of lignocaine and midazolam pharmacokinetics.

Methods  The study was conducted in four male and four female healthy volunteers, aged between 21 and 26 years, who received 1 mg kg−1 lignocaine HCl i.v. on days 1, 3, 5, 9 and 10 of the investigation. On days 5 and 10 they also received midazolam, 0.075 mg kg−1 i.v. and from days 6–10 they took erythromycin 500 mg orally, four times daily. Following administration of lignocaine and midazolam, frequent venous blood samples were obtained for determination of the concentrations of lignocaine, MEGX and midazolam.

Results  In the absence of erythromycin a statistically significant linear correlation was observed between the clearance of lignocaine and midazolam (CLmidazolam= 0.41×CLlignocaine+1.2; r2 = 0.857; P < 0.001). Erythromycin cotreatment resulted in a loss of the correlation between the two clearances (r2 = 0.39; P = 0.1). Erythromycin caused a statistically significant reduction in midazolam clearance from the original value of 3.8 to 2.5 (95% CI for the difference −2.27, −0.35) ml kg−1 min−1. Interestingly there was no significant change in the clearance of lignocaine (6.4 vs 5.8 (95% CI for the difference −2.74, −1.51) ml kg−1 min−1). Furthermore no correlation at all was observed between the MEGX‐test and lignocaine or midazolam clearances. Considering the data on day 1, 3 and 5 the intra‐individual coefficient of variation in the MEGX‐test was 45.3% at 15 min and 23.5% at 30 min, respectively.

Conclusions  It is concluded that there is a significant correlation between lignocaine and midazolam clearances but this correlation is lost after CYP3A4 inhibition by erythromycin. The MEGX‐test is of no value in assessing intra‐ and inter‐individual variability in midazolam clearance.
Beclomethasone dipropionate: absolute bioavailability, pharmacokinetics and metabolism following intravenous, oral, intranasal and inhaled administration in man
Authors: Daley‐Yates Peter T. , Price Alison C. , Sisson Jonathan R. , Pereira Adrian , Dallow Nigel
Published: March 2003

Aims  To assess the absolute bioavailability, pharmacokinetics and metabolism of beclomethasone dipropionate (BDP) in man following intravenous, oral, intranasal and inhaled administration.

Methods  Twelve healthy subjects participated in this seven‐way cross‐over study where BDP was administered via the following routes: intravenous infusion (1000 µg), oral (4000 µg, aqueous suspension), intranasal (1344 µg, aqueous nasal spray) and inhaled (1000 µg ex‐valve, metered dose inhaler). The contribution of the lung, nose and gut to the systemic exposure was assessed by repeating the inhaled, intranasal and oral dosing arms together with activated charcoal, to block oral absorption. Blood samples were collected for 24 h postdose for the measurement of BDP, beclomethasone‐17‐monopropionate (B‐17‐MP) and beclomethasone (BOH) in plasma by liquid chromatography tandem mass spectrometry.

Results  Intravenous administration of BDP (mean CL 150 l h−1, Vss 20 l, t½ 0.5 h) was associated with rapid conversion to B‐17‐MP which was eliminated more slowly (t1/2 2.7 h). In estimating the parameters for B‐17‐MP (mean CL 120 l h−1, Vss 424 l) complete conversion of BDP to B‐17‐MP was assumed. The resultant plasma concentrations of BOH were low and transient. BDP was not detected in plasma following oral or intranasal dosing. The mean absolute bioavailability (%F, 90% CI; nominal doses) of inhaled BDP was 2% (1–4%) and not reduced by coadministration of charcoal. The mean percentage F of the active metabolite B‐17‐MP was 41% (31–54%), 44% (34–58%) and 62% (47–82%) for oral, intranasal and inhaled dosing without charcoal, respectively. The corresponding estimates of nasal and lung absorption, based on the coadministration of charcoal, were < 1% and 36% (27–47%), respectively.

Conclusions  Unchanged BDP has negligible oral and intranasal bioavailability with limited absorption following inhaled dosing due to extensive (95%) presystemic conversion of BDP to B‐17‐MP in the lung. The oral and intranasal bioavailabilities of the active metabolite B‐17‐MP were high and similar, but direct absorption in the nose was insignificant. The total inhaled bioavailability of B‐17‐MP (lung + oral) was also high (62%) and approximately 36% of this was due to pulmonary absorption. Estimates of oral bioavailability and pulmonary deposition based on total BOH were approximately half those found for B‐17‐MP.
Effect of OROS® controlled‐release delivery on the pharmacokinetics and pharmacodynamics of oxybutynin chloride
Authors: Sathyan Gayatri , Chancellor Michael B. , Gupta Suneel K.
Published: March 2003

Aims  Dry mouth is a common side‐effect seen with immediate‐release oxybutynin (IR‐Oxy). Ditropan XL®[(Oxy‐XL), a controlled‐release formulation of oxybutynin chloride, is a once‐daily oral dosage form that incorporates the OROS® technology. Dry mouth as the pharmacodynamic measure was compared between Oxy‐XL and IR‐Oxy administration. The steady state stereospecific pharmacokinetics were also established for the two formulations and the kinetic‐dynamic relationship of oxybutynin was examined.

Methods  This was a randomized, repeated‐dose, double‐blind, two‐treatment, two‐period, crossover study. After a baseline assessment day, volunteers were randomly assigned to one of two treatment sequences and received 4 days of each treatment with a washout period of 7 days between treatments. The treatments were: 1) Oxy‐XL 10 mg in the morning and placebo 8 h later, and 2) IR‐Oxy 5 mg in the morning and again 8 h later. Volunteers assessed dry mouth severity subjectively using a 100 mm visual analogue scale, VAS (Baseline, treatment days 1 and 4) and objectively by collecting saliva (Baseline and treatment day 4) before dosing and every hour after the morning dose for ∼16 h. Several blood samples were collected during each treatment, with frequent sampling on day 4 to analyse for plasma R‐ and S‐oxybutynin and R‐ and S‐desethyloxybutynin concentrations.

Results  Relatively constant plasma concentrations of oxybutynin and its metabolite were seen over 24 h following Oxy‐XL administration with the degree of fluctuation being much lower (P = 0.001; 66% to 81% reduction for the various analytes) than IR‐Oxy. Compared with IR‐Oxy, Oxy‐XL yielded higher (131% and 158% for the R‐ and S‐isomer, respectively) oxybutynin and lower (62% and 78% for the R‐ and S‐isomer, respectively) desethyloxybutynin bioavailability, suggesting reduced first‐pass metabolism. Saliva output (area under the effect curve) was significantly higher [P = 0.001; 37% (95% confidence interval: 24, 51%)] with Oxy‐XL than with IR‐Oxy and, accordingly, dry mouth severity (VAS) integrated over the day was significantly lower with Oxy‐XL. The decrease in saliva output and the consequent increase in dry mouth severity correlated with the metabolite R‐desethyloxybutynin concentration, and no apparent relationship was observed with the R‐oxybutynin concentration. This suggests that the metabolite may contribute to the dry mouth. Therefore, the reduction in metabolite exposure with Oxy‐XL may be a possible explanation for the observed decrease in dry mouth severity with OXY‐XL compared with IR‐Oxy.

Conclusions  Oxy‐XL maintains relatively constant plasma drug and metabolite concentrations and minimizes first‐pass metabolism of oxybutynin. The metabolite appears to contribute to dry mouth associated with oxybutynin, and following Oxy‐XL metabolite exposure is reduced compared with IR‐Oxy. Consequently less dry mouth was observed with Oxy‐XL as compared with IR‐Oxy.
The glucuronidation of mycophenolic acid by human liver, kidney and jejunum microsomes
Authors: Bowalgaha Kushari , Miners John O.
Published: March 2003

Aims  To estimate the relative contribution of liver, kidney and jejunum to MPA elimination via glucuronidation from in vitro kinetic data.

Methods  The kinetics of MPA glucuronidation by human liver, kidney and jejunum microsomes were characterized. Mycophenolic acid glucuronide (MPAG) concentrations in microsomal incubations were determined using a specific h.p.l.c. procedure. Non‐specific microsomal binding of MPA was excluded using an equilibrium dialysis approach.

Results  Microsomes from all three tissues catalysed the conversion of MPA to MPAG. Mean microsomal intrinsic clearances for MPAG formation by liver, kidney and jejunum microsomes were 46.6, 73.5 and 24.5 µl (min mg)−1, respectively. When extrapolated to the whole organ, however, hepatic intrinsic clearance was 21‐ and 38‐fold higher than the respective intrinsic clearances for kidney and small intestine.

Conclusions  The data suggest that the liver is the organ primarily responsible for the systemic clearance of MPA, with little contribution from the kidney, and that the small intestine would be expected to contribute to first‐pass extraction to a minor extent only.
Pharmacogenetics of asthma
Authors: Fenech A. , Hall I. P.
Published: March 2003

Recent advances in the extent of knowledge regarding interindividual genetic variation in drug treatment targets and drug metabolizing enzymes has resulted in studies designed to assess the contribution of genetic variability to treatment response in a range of diseases. This review describes the current state of knowledge of genetic variability in key airway targets important in the treatment of asthma. Whilst the genes coding for some key treatment targets contain little polymorphic variation (e.g. the muscarinic M2 and M3 receptors) other genes whose products are important targets in the treatment of asthma contain extensive genetic variation. The best examples of the latter are the β2‐adrenoceptor and the 5‐lipoxygenase genes. Genetic variability in both of these genes may account in part for interindividual variability in treatment response. Finally, a number of key targets within the airways remain to be adequately screened for polymorphic variation.
Correlation and predictive performances of saliva and plasma nicotine concentration on tobacco withdrawal‐induced craving
Authors: Teneggi Vincenzo , Squassante Lisa , Iavarone Laura , Milleri Stefano , Bye Alan , Gomeni Roberto
Published: March 2003

Aims
To investigate whether saliva is a useful alternative to plasma for routine monitoring of nicotine and evaluate the predictive performances of saliva and plasma concentration on craving estimated by a Tiffany Questionnaire on Smoking Urge‐Brief Form.

Methods
Thirteen healthy smokers were enrolled in a randomized, two period, crossover trial. Linear and power models were evaluated to predict the plasma nicotine concentrations from the saliva measurements, whereas a population PK/PD indirect response model was used to predict craving using either saliva or plasma nicotine concentration as the independent variable.

Results
The   results   of   the   analysis   revealed   that   the   power   model   was   preferred over the   linear   one   The   bias   on   the   predicted   plasma   concentrations   was of 0.47 ng ml−1 with a 95% confidence interval of [‐0.57, 1.52] and a precision of 5.68 ng ml−1. The placebo effect model was initially fitted to data, then the indirect response approach (with inhibition in kin) was used to model the craving scores using plasma and saliva nicotine concentrations as independent variables. The two indirect response PK/PD models based on saliva and plasma nicotine concentrations, adequately described the onset, extent, and duration of craving. The maximal inhibition Imax was 0.722 and 1 for saliva and plasma concentrations while the estimated nicotine concentrations giving 50% of the maximal inhibition were 269 ng ml−1 and 24.3 ng ml−1 for saliva and plasma, respectively.

Conclusions
A good correlation between plasma and saliva nicotine concentrations has been found using a power model. Comparable values of bias and precision on the model‐predicted craving indicate that plasma and saliva concentration can equally well be used to predict the onset of tobacco withdrawal induced craving. Analysis of saliva definitely offers a potentially more attractive way to assess nicotine concentration values, as samples can be collected easily and noninvasively. In addition, saliva sampling avoids the pain and discomfort involved in venepuncture. In studies that assess psychological measures, such as subjective mood, blood collection could present a possible confounding factor because of the anxiety and pain that accompanies it. For these reasons saliva can reasonably be considered as the ideal sampling site for all clinical studies conducted for the evaluation of the potential activity of drugs on nicotine deprivation symptoms.
Types of carbonate platforms: a genetic approach
Authors: Pomar L.
Published: March 2003

ABSTRACTMany types of carbonate platforms have been described, from homoclinal ramps to rimmed shelves and a full spectrum of variations in between; the distinction between these different types can be problematic. Nevertheless, classification of carbonate platforms is not just a semantic or academic issue. For example, it is clearly important for the accurate interpretation of seismic images of facies geometry and for assessing the potential of stratigraphic traps. Even though predictive efficiency of conceptual models depends on the degree of comprehension of the genetic factors controlling depositional profiles and the distribution of facies belts, current models for classification of carbonate platforms are basically descriptive and mainly based on depositional profile, size, and attachment to or detachment from a landmass.

A genetic approach considers the variability of depositional profiles among carbonate platforms as a function of the type of sediment that was produced (basically grain size), the locus of sediment production, and the hydraulic energy. Three groups of carbonate‐producing biota may be distinguished according to their dependence upon light: (1) euphotic (good light) in shallow, wave‐agitated areas; (2) oligophotic (poor light) in deeper, commonly non‐wave‐agitated areas; and (3) photo‐independent biota in all water‐depth ranges.

Several platform types in wave‐dominated seas can be considered in relation to genetic factors, even when simplifying the many possible scenarios. Euphotic framework‐producing biota create rimmed shelves similar to modern reef platforms. Soft‐substrate‐dwelling biota, which produce gravel‐sized carbonate in the shallow euphotic zone, create flat‐topped open shelves. Oligophotic gravel‐producing biota, such as some larger foraminifera and red algae, generate distally steepened ramps. Mud‐dominated carbonate production, in either euphotic or oligophotic zones, generate homoclinal ramps. Carbonate production dominated by photo‐independent biota (crinoids, sponges, bryozoans, etc.) above wave base give rise to open shelves or ramps, depending upon grain size, but may produce mounds if carbonate production occurs below the base of wave/current sweeping.
Neuropeptide Y alters sedation through a hypothalamic Y1‐mediated mechanism
Authors: Naveilhan Philippe , Canals Josep M. , Valjakka Antti , Vartiainen Jukka , Arenas Ernest , Ernfors Patrik
Published: March 2003

Neuropeptide Y (NPY) has been reported to profoundly influence and regulate brain circuits involved in a number of behaviours, like anxiety, alcohol intake, pain and energy homeostasis. Here we show that NPY increases sedation induced by different types of anaesthetics through interactions with the Y1 receptor. Consistently, in Y1–/– (homozygote knockout) mice NPY does not potentiate the pentobarbital‐induced sedation. Similar results were obtained for avertin but not for ketalar‐ (NMDA antagonist) induced sedation. Local microinjection of NPY exhibited the strongest potentiating effect on pentobarbital‐induced sedation in the posterior hypothalamic area and Y1 expression was found in the dorsal‐premammillary and medial part of medial mammillary nuclei. These results show that Y1 is essential for NPY‐induced enhancement of sedation and place this activity of NPY in the posterior hypothalamic area, a region of the brain previously implicated in the regulation of the wake–sleep cycle.
Design and characterization of a DNA‐encoded, voltage‐sensitive fluorescent protein
Authors: Sakai Rieko , Repunte‐Canonigo Vez , Raj Christopher D. , Knöpfel Thomas
Published: March 2003

Optical imaging of electrical activity has been suggested as a promising approach to investigate the multineuronal representation of information processing in brain tissue. While considerable progress has been made in the development of instrumentation suitable for high‐speed imaging, intrinsic or extrinsic dye‐mediated optical signals are often of limited use due to their slow response dynamics, low effective sensitivity, toxicity or undefined cellular origin. Protein‐based and DNA‐encoded voltage sensors could overcome these limitations. Here we report the design and generation of a voltage‐sensitive fluorescent protein (VSFP) consisting of a voltage sensing domain of a potassium channel and a pair of cyan and yellow emitting mutants of green fluorescent protein (GFP). In response to a change in transmembrane voltage, the voltage sensor alters the amount of fluorescence resonance energy transfer (FRET) between the pair of GFP mutants. The optical signals respond in the millisecond time‐scale of fast electrical signalling and are large enough to allow monitoring of voltage changes at the single cell level.
Cysteinyl leukotriene‐dependent interleukin‐5 production leading to eosinophilia during late asthmatic response in guinea‐pigs
Authors: Nabe T. , Yamashita K. , Miura M. , Kawai T. , Kohno S.
Published: March 2003

Background Allergic airway eosinophilia is suppressed by cysteinyl leukotriene (CysLT) receptor (CysLT1 receptor) antagonists in several species including humans and guinea‐pigs, suggesting that CysLTs are directly or indirectly involved in induction of the response.

Objective We examined the effect of CysLT antagonists (pranlukast and MCI‐826) on antigen inhalation‐induced eosinophilia in peripheral blood and lung, and on IL‐5 activity in serum during late increase of airway resistance (late asthmatic response, LAR) in sensitized guinea‐pigs.

Methods Guinea‐pigs inhaled ovalbumin (OVA) + Al(OH)3 and OVA mists alternately for sensitization and challenge, respectively, once every 2 weeks. At the fifth challenge, the effects of CysLT antagonists and an anti‐IL‐5 antibody (TRFK‐5) on the occurrence of LAR, and blood and lung eosinophilia, which appeared at 5 h after challenge, were examined. The time‐course of IL‐5 activity in the serum after the challenge was evaluated by measuring in vitro‘eosinophil survival prolongation activity’. The influence of CysLT antagonists on IL‐5 activity was assessed.

Results CysLT antagonists and TRFK‐5 completely abolished blood and lung eosinophilia. LAR was suppressed by both MCI‐826 and TRFK‐5 by 40–50%. Sera obtained from sensitized, challenged animals 3 h and 4 h after challenge induced an obvious prolongation of eosinophil survival. The activity of the sera was completely neutralized by prior exposure to TRFK‐5, suggesting that it reflected IL‐5 activity. Increased IL‐5 activity in the serum was inhibited by both pranlukast and MCI‐826 by over 90%.

Conclusions CysLTs produced after antigen provocation sequentially induced IL‐5 production from some immune component cells via CysLT1 receptor activation. Thus, it is likely that CysLTs indirectly cause antigen‐induced eosinophilia through IL‐5 production.