bluuebunny/crossref_metadata_2025 huggingface dataset:
Modern Social Democracy and European Welfare State Reform
Authors: Clasen Jochen
Published: July 2002

The European political landscape in the 1990s was characterised by centre-left parties returning to power in several countries after long periods in opposition. Following intensive internal debates over policy direction and policy revision, once back in power some centre-left governments in some countries have used terms such as the ‘third way’ and ‘new social democracy’, or the ‘Neue Mitte’ as an indication that contemporary policies should be seen as distinct from those pursued by both previous social democratic administrations and neo-liberal governments in the interim (e.g. Gamble and Wright, 1999; White, 2001). What, if anything, exactly constitutes the ‘third way’ has been a matter of considerable debate, as has been the question of how far traditional social democratic values and aspirations, such as solidarity and equality, are still relevant within ‘third way’ policies. More than previously, modern social democratic policy is geared towards reducing non-wage labour costs, fostering private forms of social protection, such as funded pensions, intensifying labour market integration and subsidising low-skilled jobs, but also incorporating new social risks and new social needs (Vandenbrouke, 2001).
Social Science and the Evidence-based Policy Movement
Authors: Young Ken , Ashby Deborah , Boaz Annette , Grayson Lesley
Published: July 2002

There is a growing interest in ‘evidence-based policy making’ in the UK. However, there remains some confusion about what evidence-based policy making actually means. This paper outlines some of the models used to understand how evidence is thought to shape or inform policy in order to explore the assumptions underlying ‘evidence-based policy making.’ By way of example, it considers the process of evidence seeking and in particular the systematic review as a presumed ‘gold standard’ of the EBP movement. It highlights some of the opportunities and challenges represented in this approach for policy research. The final part of the paper outlines some questions of capacity that need to be addressed if the social sciences are to make a more effective contribution to policy debate in Britain.
Studies on the autoxidation and sensitizing capacity of the fragrance chemical linalool, identifying a linalool hydroperoxide
Authors: Sköld Maria , Börje Anna , Matura Mihaly , Karlberg Ann‐Therese
Published: July 2002

Fragrances are among the most common causes of allergic contact dermatitis. The two monoterpenes linalool and d‐limonene are the most frequently incorporated fragrance chemicals in scented products. Previous studies on d‐limonene show that this monoterpene oxidizes on air exposure (autoxidation) and that allergenic oxidation products are formed. Due to structural similarities, linalool might also form allergenic oxidation products on air exposure. The aim of the present study was to study the autoxidation of linalool and to investigate the sensitizing potential of linalool before and after air exposure. Linalool was oxidized for 10 weeks and gas chromatographic analyses showed that the content of linalool decreased to about 80%. The chromatograms revealed the formation of other compounds during oxidation. One of the major oxidation products was isolated and identified as 7‐hydroperoxy‐3,7‐dimethyl‐octa‐1,5‐diene‐3‐ol. This substance is, to the best of our knowledge, described for the first time. In sensitization studies in guinea pigs, linalool of high purity gave no reactions, while linalool that had been oxidized for 10 weeks sensitized the animals. It is concluded that autoxidation of linalool is essential for its sensitizing potential.
Tacrolimus enhances irritation in a 5‐day human irritancy in vivo model
Authors: Fuchs M. , Schliemann‐Willers S. , Heinemann C. , Elsner P.
Published: July 2002

Tacrolimus (FK 506) is a macrolide discovered in 1984 as a metabolic product of Streptomyces tsukabaensis. It has been used successfully in treating atopic dermatitis, allergic contact dermatitis, lichen planus mucosae and pyoderma gangrenosum. In the present study, we evaluated the anti‐inflammatory activity of FK 506 in 2 human skin inflammation models. FK 506 as Protopic® cream was tested (i) in a 4‐day repetitive irritation test with 2 × daily application of sodium lauryl sulphate (SLS), and (ii) in a UVB erythema model. The effect was evaluated visually and quantified by non‐invasive bioengineering methods, namely chromametry and tewametry (TEWL). When FK 506 was applied 30 min after SLS irritation, an increased inflammation in comparison to controls was observed with all 3 methods, with only the TEWL data reaching statistical significance. 1 × daily application of FK 506 for 5 days, starting at the end of the 4‐day irritation period, was without any effect. Similarly, no effect of FK 506 was seen in the UVB model. In conclusion, FK 506 was shown to enhance experimentally induced irritant contact dermatitis and not to accelerate healing of irritant contact dermatitis and UVB erythema.
Electrophysiological and neuropsychological tests for the diagnosis of subclinical hepatic encephalopathy and prediction of overt encephalopathy
Authors: Saxena Nandini , Bhatia Manvir , Joshi Yogendra K. , Garg Pramod K. , Dwivedi Sada Nand , Tandon Rakesh K.
Published: July 2002

Abstract:  Background: Subclinical hepatic encephalopathy (SHE) features in 30–84% of patients with cirrhosis of the liver. Its clinical significance with regards to progression to overt encephalopathy has however, not been established.

Aims: The present study was conducted (i) to compare the diagnostic usefulness of neuropsychological tests with that of electrophysiological (EP) tests in detection of SHE, and (ii) to examine the natural course of SHE.

Methods: Seventy‐five‐nonencephalopathic cirrhotics (11 females, 64 males; mean (± SD) age 43.6 (± 11.7) years; mean (± SD) education 11(± 3) years) were studied using a battery of tests for intelligence and memory, the number connection test (NCT), and EP tests viz. electroencephalogram (EEG) and auditory P300 event related potentials (P3ERP). All the patients were followed up for a period of 6 months to 2 years for development of overt encephalopathy.

Results: Thirty‐five out of 75(47%) patients were diagnosed to have SHE based on at least one abnormal test result. The P3ERP latencies detected SHE in maximum number of patients (23%) followed by EEG (21%). Nearly 59% of patients with SHE progressed to overt encephalopathy within a mean duration of 4 months. Multivariate analysis showed that prior episode of encephalopathy (RR = 6.3; 95% CI = 2.0–19.7), abnormality on EEG (RR = 7.5; 95% CI = 2.2–25.3), abnormal performance on psychometric battery of tests (RR = 35.2; 95% CI = 4.3–287.3), occurrence of gastrointestinal bleed (RR = 19.3; 95% CI = 4.1–88.9), occurrence of dehydration (RR = 10.7; 95% CI = 2.5–45.4) and infection (RR = 11.4; 95% CI = 2.0–64.4) had significantly higher risk for development of overt encephalopathy.

Conclusions: EP methods were more sensitive in detection of SHE. Amongst all the tests used, presence of only an abnormal EEG was significantly associated with development of overt encephalopathy along with the precipitating factors.
Correlation of HBV DNA levels in serum and liver of chronic hepatitis B patients with cirrhosis
Authors: Lee Cha Ze , Huang Guan Tarn , Yang Pei Ming , Sheu Jin Chuan , Lai Ming Yang , Chen Ding Shinn
Published: July 2002

Abstract:  Aim: We sought to evaluate whether serum HBV DNA levels correlates with the liver free HBV DNA levels in chronic hepatitis B.

Methods: Thirty‐three consecutive chronic hepatitis B patients with cirrhosis were included in this study. Twenty cases had detectable serum HBV DNA (> 1.8 pg/ml). All had detectable free liver HBV DNA.

Results: There was a strong correlation between the serum and liver HBV DNA levels (P = 0.0018, r = 0.717). Thirteen cases had undetectable serum HBV DNA. Among them, six cases still had detectable liver free HBV DNA. Eight cases were HBeAg‐positive. Among them, seven cases were positive for both serum and liver HBV DNA, and one case was negative for both serum and liver HBV DNA. Twenty‐five cases were HBeAg‐negative and anti‐HBe‐positive. Among them, 13 cases were positive for serum HBV DNA, and 19 cases were positive for liver HBV DNA. No significant difference was noted for positivity of serum HBV DNA or liver free HBV DNA between HBeAg‐positive and HBeAg‐negative groups. The level of serum HBV DNA(491 ± 772 pg/ml versus 203 ± 447 pg/ml, p = 0.07) and liver free HBV DNA (33 ± 81 pg/µg versus 6 ± 15 pg/µg, p = 0.13) was also not statistically different.

Conclusion: In conclusion, serum HBV DNA levels is strongly correlated with liver HBV DNA levels in chronic hepatitis B with cirrhosis. Liver free HBV DNA can still be detected in about half of the cirrhotic patients with undetectable serum HBV DNA. Serum HBeAg is not a good predictor of serum or liver HBV DNA levels in cirrhotic patients.
Expression of p57/Kip2 protein in extrahepatic bile duct carcinoma and intrahepatic cholangiocellular carcinoma
Authors: Ito Yasuhiro , Takeda Tsutomu , Sasaki Yo , Sakon Masato , Yamada Terumasa , Ishiguro Shingo , Imaoka Shingi , Tsujimoto Masahiko , Monden Morito , Matsuura Nariaki
Published: July 2002

Abstract:  Background: Evaluation of the biological character of carcinomas requires understanding of cell cycle regulators. P57 (Kip2) belongs to the Cip/Kip family and is known to be one of the universal negative regulators of cell cycle.

Methods: In the present study, therefore, we investigated p57 expression in 37 extrahepatic bile duct carcinomas (BDC) and 28 intrahepatic cholangiocellular carcinomas (CCC).

Results: The average p57 labeling index (LI) in BDC and CCC were 60.8 ± 7.9 and 58.6 ± 18.6, respectively, which were significantly lower (p = 0.0008 and p = 0.0408, respectively) than those in normal duct epithelia (73.1 ± 7.9, 70.4 ± 8.2). p57 LI was significantly lower in BDC and CCC cases with biological aggressive phenotypes such as poor differentiation (p = 0.0260 and p = 0.0069), lymph node metastasis (p = 0.0274 and p = 0.0214), high Ki‐67 LI (p = 0.0164 and p = 0.0343) and cyclin D1 overexpression (p = 0.0359 and p = 0.0255).

Conclusion: These findings suggest that decreased p57 expression is related to the increased activity of cell proliferation and also the progression of these carcinomas.
Relationship between protein kinase C alterations and nitric oxide overproduction in cirrhotic rat aortas
Authors: Tazi KA , Barrière E , Moreau R , Poirel O , Lebrec D. , Tazi Khalid A. , Barrière Eric , Moreau Richard , Poirel Odile , Lebrec Didier
Published: July 2002

Abstract:  Background: Although nitric oxide (NO) overproduction and protein kinase C (PKC) alterations may play a role in systemic haemodynamic changes in cirrhotic rat aortas, the relationship between NO synthase (NOS) hyperactivation and PKC hypoactivation is unknown. Therefore, the relationships between NOS and PKC activities were studied in cirrhotic rat aortas.

Methods: The effects of NOS inhibition by Nw‐nitro‐L‐arginine (LNNA) on the contractile response to phorbol myristate acetate (PMA), a PKC activator, were studied. The effects of NOS inhibition and those of S‐nitroso‐N acetyl‐DL‐penicillamine (SNAP), an NO donor, on PKC activity were also evaluated. The effects of PKC activation and inhibition on total NOS and inducible NOS (iNOS) activities were measured. Nitric oxide synthase inhibition caused an increase in PMA‐induced contraction and an increase in PKC activity in cirrhotic rat aortas. S‐nitroso‐N acetyl‐DL‐penicillamine induced downregulation of PKC activity. Total basal aortic NOS activity was significantly higher in cirrhotic rats than in control rats and activation of PKC by PMA induced a decrease in total aortic NOS activity. Protein kinase C downregulation caused an increase in both total aortic NOS and iNOS activities only in control rats, whereas only iNOS activity increased in cirrhotic rats.

Conclusion: In cirrhotic rat aortas, NO overproduction plays a role in the decreased PKC activation that leads to reduced aortic contraction. Overactivation of aortic NOS in cirrhotic rats may be because of , in part, the reduced PKC activity.
Pressor and pulmonary responses to ET‐1(1–31) in guinea‐pigs
Authors: Honoré Jean‐Claude , Plante Mirco , Bkaily Ghassan , Rae Giles A , D'Orléans‐Juste Pedro
Published: July 2002

Endothelin‐1(1–31) (ET‐1(1–31); 0.25 to 4 nmol kg−1; i.v.) induced, in the guinea‐pig, graded increases in MAP and an indomethacin‐sensitive enhancement of pulmonary insufflation pressure (PIP). At all doses, ET‐1(1–31) induced a monophasic pressor response, except at 4 nmol kg−1, which caused a rapid and transient response (first phase: over first 10 min after injection) followed by a more slowly‐developing and sustained (second phase: between 10 and 45 min after injection) increase in MAP. ET‐1(1–31) was 4 to 10 fold less potent than ET‐1 on PIP responses.

Phosphoramidon (5 and 10 mg kg−1) reduced both pressor and PIP effects of ET‐1(1–31). Thiorphan (0.25 and 2.5 mg kg−1) did not affect the pressor responses to ET‐1(1–31) although its PIP effects were markedly reduced by the NEP inhibitor. A selective endothelin‐converting enzyme (ECE) inhibitor, CGS 35066 (1 mg kg−1), significantly reduced the second phase pressor response and increase in PIP triggered by ET‐1(1–31).

The second (but not the first) pressor phase of ET‐1(1–31) (4 nmol kg−1) was markedly reduced by BQ‐123 (selective ETA antagonist), whereas the increase of PIP was significantly reduced by BQ‐788 (selective ETB antagonist). Co‐administration of BQ‐123 plus BQ‐788 abolished ET‐1(1–31)‐induced increase in PIP, but blockade of the second pressor phase afforded by BQ‐123 was now reversed.

In guinea‐pig isolated perfused lungs, ET‐1(1–31) (50 nM) induced the release of prostacyclin and thromboxane A2, which was inhibited by BQ‐788 (5 nM) or thiorphan (25 μM), but not BQ‐123 (1 μM).

These results suggest that ET‐1(1–31) enhances MAP. Its sustained, but not transient, pressor effects are mediated via ETA receptor activation. Furthermore, ET‐1(1–31) increases airway resistance in vivo and triggers prostacyclin and thromboxane A2 release from perfused lungs predominantly via ETB receptor activation. ET‐1(1–31) failed to display any selectivity of action towards either ETA or ETB receptors in these models.

We suggest that, in order to raise MAP, ET‐1(1–31) requires conversion to ET‐1, predominantly by ECE and to a lesser extent neutral endopeptidase 24.11, whereas the reverse holds true regarding its pharmacological effects in airways.

British Journal of Pharmacology (2002) 136, 819–828. doi:10.1038/sj.bjp.0704782
Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3
Authors: Hayer‐Zillgen Martina , Brüss Michael , Bönisch Heinz
Published: July 2002

Organic cation transporters (OCTs) are involved in the elimination of monoamines and cationic xenobiotics. To examine whether some cell lines express several different OCTs, we investigated seven human cell lines for the mRNA expression pattern of the human (h) transporters hOCT1, hOCT2 and hOCT3. hOCT1 mRNA was found in all cell lines, six additionally expressed hOCT3 and only two cell lines contained all three hOCTs.

Among the three OCTs only for the OCT3 (also designated as ‘uptake2’ or ‘extraneuronal monoamine transporter’) ‘selective’ inhibitors are described in the literature. The affinities of the OCT3 inhibitors for the other two OCTs are largely unknown. Therefore, we compared the potencies of eight compounds as inhibitors of hOCT‐mediated uptake of the organic cation [3H]‐1‐methyl‐4‐phenylpyridinium ([3H]‐MPP+) in human embryonic kidney 293 (HEK293) cells stably expressing hOCT1, hOCT2 or hOCT3. Decynium‐22 inhibited hOCT3 with 10 fold higher potency than hOCT1 and hOCT2. Corticosterone was about 100 fold more potent as inhibitor of hOCT3 than of hOCT1 or hOCT2, and O‐methylisoprenaline (OMI) inhibited almost exclusively hOCT3. Progesterone and β‐Oestradiol preferentially inhibited hOCT3 and hOCT1, whereas prazosin was a potent inhibitor of hOCT1 and hOCT3. Phenoxybenzamine (PbA) inhibited with about equal apparent potency all three hOCTs, whereas the PbA derivative SKF550 ((9‐fluorenyl)‐N‐methyl‐β‐chloroethylamine) preferentially inhibited hOCT3 and hOCT2.

PbA reversibly inhibited hOCT1 and irreversibly hOCT2 and hOCT3; SKF550 also irreversibly inhibited hOCT3 but hOCT2 in a reversible manner.

These compounds enable a functional discrimination of the three hOCTs: hOCT1 is selectively inhibited by prazosin, reversibly inhibited by PbA and it is not sensitive to inhibition by SKF550 and OMI; hOCT2 is reversibly inhibited by SKF550, irreversibly by PbA and not by prazosin, β‐oestradiol and OMI, whereas hOCT3 is selectively inhibited by corticosterone, OMI and decynium22.

British Journal of Pharmacology (2002) 136, 829–836. doi:10.1038/sj.bjp.0704785
FcεRI cross‐linking‐induced actin assembly mediates calcium signalling in RBL‐2H3 mast cells
Authors: Oka Tatsuya , Sato Koichi , Hori Masatoshi , Ozaki Hiroshi , Karaki Hideaki
Published: July 2002

To determine the role of actin assembly in the Ca2+ signalling of mast cells activated by cross‐linking of FcεRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization.

In the RBL‐2H3 cells, F‐actin content was increased by sensitization with anti‐dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca2+ ([Ca2+]i); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments.

In the IgE‐sensitized RBL‐2H3 cells, DNP‐human serum albumin (DNP‐HSA) augmented actin assembly. DNP‐HSA also increased the production of IP3, [Ca2+]i and degranulation. Cytochalasin D enhanced all of these DNP‐HSA‐induced effects.

In a Ca2+‐free solution, DNP‐HSA induced a transient increase in [Ca2+]i, and this increase was accelerated by cytochalasin D. After cessation of the DNP‐HSA‐induced Ca2+ release, the re‐addition of Ca2+ induced a sustained increase in [Ca2+]i through capacitative Ca2+ entry (CCE), and this increase was enhanced by cytochalasin D.

The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C.

In contrast, neither the Ca2+ release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D.

These results suggest that actin de‐polymerization stimulates the FcεRI‐mediated signalling to augment the release of Ca2+ from the endoplasmic reticulum in RBL‐2H3 cells.

British Journal of Pharmacology (2002) 136, 837–846. doi:10.1038/sj.bjp.0704788
Investigation of neurotransmission in vas deferens from α2A/D‐adrenoceptor knockout mice
Authors: Cleary Linda , Vandeputte Catherine , Docherty James R
Published: July 2002

We have investigated pre‐ and post‐junctional responsiveness in vas deferens from wild‐type and α2A/D‐adrenoceptor knockout mice. The response to a single stimulus was not significantly different between wild‐type and knock‐out mice. The isometric contraction to 10 Hz stimulation for 4 s was significantly larger in vas deferens from knockout as compared with wild‐type.

The maximum potentiation of 10 Hz stimulation‐evoked contractions by yohimbine was to 206.2±38.0% of control in wild‐type but to 135.8±13.6% of control in knockout. The α2A/D‐adrenoceptor selective antagonist BRL 44408 significantly increased the 10 Hz stimulation‐evoked contraction in wild‐type but not knockout, and the reverse was true for the α2C‐adrenoceptor selective antagonist spiroxatrine. The α2B‐adrenoceptor antagonist imiloxan had no effect on the evoked contraction except at high concentrations, and only in wild‐type. Following cocaine (3 μM) and BRL 44408 (1 μM), 10 Hz responses were similar in shape and maximum between wild‐type and knock‐out.

The α2‐adrenoceptor agonist xylazine virtually abolished the early component of the contraction to 10 Hz stimulation in the presence of nifedipine (10 μM) in vas deferens from knockout mice in a way consistent with a change of receptor subtype but without clear evidence for a reduced receptor number. However, the late component of the contraction to 10 Hz stimulation was significantly potentiated by xylazine in tissues from knock‐out mice.

It is concluded that, although non‐α2A/D‐adrenoceptors replace α2D‐adrenoceptors in this knockout, the α2‐adrenoceptor agonist and antagonist data are contradictory. The antagonist data suggest a major loss of prejunctional α2‐adrenoceptors, but this is not necessarily supported by the agonist data.

British Journal of Pharmacology (2002) 136, 857–864. doi:10.1038/sj.bjp.0704791
Isolation and characterization of the monkey UGT2B30 gene that encodes a uridine diphosphate-glucuronosyltransferase enzyme active on mineralocorticoid, glucocorticoid, androgen and oestrogen hormones
Authors: GIRARD Caroline , BARBIER Olivier , TURGEON David , BÉLANGER Alain
Published: July 2002

The present study reports the genomic organization and the characterization of a novel cynomolgus monkey UDP-glucuronosyltransferase (UGT) enzyme, UGT2B30. UGT enzymes are microsomal proteins that catalyse the transfer of the glucuronosyl group from UDP-glucuronic acid (UDPGA) to a wide variety of lipophilic compounds, namely hormonal steroids. The 15kb UGT2B30 gene amplified by PCR showed a genomic organization similar to those encoding UGT2B human enzymes. The cDNA encoding UGT2B30 was isolated from a cynomolgus monkey prostate cDNA library, and the deduced amino acid sequence showed an identity of 94% with UGT2B19, a monkey isoform previously characterized. Stable expression of UGT2B30 protein in human kidney 293 (HK293) cells was assessed by Western-blot analysis and its conjugating activity was screened using 39 potential substrates. The UGT2B30 enzyme is active on many compounds of different classes, including testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol, androsterone, oestradiol, tetrahydroaldosterone and tetrahydrocortisone, with glucuronidation efficiencies (Vmax/Km ratios) ranging from 0.6 to 8.8μl·min−1·mg of protein−1. Reverse-transcriptase-PCR analysis revealed that the UGT2B30 transcript is expressed in several tissues, including prostate, testis, mammary gland, kidney, adrenals and intestine. The relative activity of UGT2B30 in comparison with other simian UGT2B isoforms, as well as its large variety of substrates, strongly suggest that this enzyme is essential to inactivation of several steroids.
Function of the 90-loop (Thr90–Glu100) region of staphylokinase in plasminogen activation probed through site-directed mutagenesis and loop deletion
Authors: RAJAMOHAN Govindan , DAHIYA Monika , MANDE Shekhar C. , DIKSHIT Kanak L.
Published: July 2002

Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr90—Glu100) for the docking of the substrate PG to the SAK—Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr92—Glu100) from the SAK 90-loop, resulted in ≈60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKΔ90) and Pm. The preformed activator complex of SAKΔ90 with Pm, however, displayed a 50–60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK—Pm complex displayed ∼50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKΔ90. Lysine residues (Lys94, Lys96, Lys97 and Lys98) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAKK97A and SAKK98A exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the Km values of PG towards the activator complex of these two SAK mutants were 4–6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys97 and Lys98 residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK—Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.
Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids
Authors: WADUM Majken C.T. , VILLADSEN Jens K. , FEDDERSEN Søren , MØLLER Rikke S. , NEERGAARD Thomas B.F. , KRAGELUND Birthe B. , HØJRUP Peter , FÆRGEMAN Nils J. , KNUDSEN Jens
Published: July 2002

Long-chain acyl-CoA esters are key metabolites in lipid synthesis and β-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525nm respectively, in the absence of ligand (excitation 387nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460nm upon binding of C14—C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (Kd = 0.6–1.7nM). FACI-53 showed a high fluorescence yield for C8—C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.
Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium
Authors: IGARASHI Kiyohiko , MOMOHARA Ikuo , NISHINO Takeshi , SAMEJIMA Masahiro
Published: July 2002

The pre-steady-state kinetics of inter-domain electron transfer in the extracellular flavocytochrome cellobiose dehydrogenase from Phanerochaete chrysosporium was studied using various values of pH and substrate concentration. Monitoring at the isosbestic point of each prosthetic group indicated that the reductive half-reactions of flavin and haem were biphasic and monophasic respectively. When the observed rates of the flavin and haem reactions were plotted against substrate concentration, the behaviour of the second phase of the flavin reduction was almost identical with that of haem reduction at all substrate concentrations and pH values tested, suggesting that the formation of flavin semiquinone and haem reduction involve the same electron transfer reaction. Although flavin reduction by cellobiose was observed in the range of pH3.0–7.0, the velocity of the next electron transfer step decreased with increase of pH and was almost zero above pH6.0. The second phase of flavin reduction and the haem reduction were inhibited similarly by high concentrations of the substrate, whereas the first phase of flavin reduction showed a hyperbolic relation to the cellobiose concentration. Increase in pH enhanced the substrate inhibition of haem reduction but not the initial flavin reduction. Moreover, the dissociation constant Kd of flavin reduction and the substrate inhibition constant Ki of haem reduction decreased similarly with an increase of pH. From these results, it is evident that binding of cellobiose to the active site inhibits electron transfer from flavin to haem.
Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human
Authors: FUKUDA Mitsunori , OGATA Yukie , SAEGUSA Chika , KANNO Eiko , MIKOSHIBA Katsuhiko
Published: July 2002

Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIα), the original Syt VII, and two minor isoforms (Syt VIIβ and Syt VIIγ), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIα. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIα (65kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII—green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.
Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme
Authors: GUO Jianxia , ZIMNIAK Ludwika , ZIMNIAK Piotr , ORCHARD John L. , SINGH Shivendra V.
Published: July 2002

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1—mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67—78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.
Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis
Authors: GURCHA Sudagar S. , BAULARD Alain R. , KREMER Laurent , LOCHT Camille , MOODY D.Branch , MUHLECKER Walter , COSTELLO Catherine E. , CRICK Dean C. , BRENNAN Patrick J. , BESRA Gurdyal S.
Published: July 2002

Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis.
Activation of protein kinase CK2 is an early step in the ultraviolet B-mediated increase in interstitial collagenase (matrix metalloproteinase-1; MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts
Authors: BRENNEISEN Peter , WLASCHEK Meinhard , SCHWAMBORN Edith , SCHNEIDER Lars-Alexander , MA Wenjian , SIES Helmut , SCHARFFETTER-KOCHANEK Karin
Published: July 2002

Enhanced expression of matrix metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in skin fibroblasts and subsequent damage of dermal connective tissue in the context of sun-induced premature aging and skin tumour progression is causally linked to UVB irradiation. Here, we were interested in identifying components of the complex signal-transduction pathway underlying UVB-mediated up-regulation of these delayed UV-responsive genes and focused on components maximally activated early after irradiation. A 2.3-fold increase in protein kinase CK2 activity was measured at 20–40min after low-dose UVB irradiation (at 10mJ/cm2) of dermal fibroblasts. This UVB-mediated increase in CK2 activity was abrogated by pharmacological approaches using non-toxic concentrations of the CK2 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). Preincubation of fibroblasts with DRB prior to UVB irradiation lowered MMP-1 by 49–69% and MMP-3 protein levels by 55–63% compared with UVB-irradiated controls. By contrast, the CK2 inhibitor did not affect the UVB-triggered transcription of MMPs. Furthermore, UVB irradiation of fibroblasts overexpressing a kinase-inactive mutant of CK2 (CK2α-K68A-HA) resulted in lowering of the protein levels of MMP-1 by 25% and MMP-3 by 22% compared with irradiated fibroblasts transfected with the vector control. This reduction in MMP protein levels correlated with the transfection efficiency. Taken together, we describe a novel aspect of protein kinase CK2, namely its inducible activity by UVB irradiation, and provide evidence that CK2 is an early mediator of the UVB-dependent up-regulation of MMP-1 and MMP-3 translation, whereas their major tissue inhibitor of matrix metalloproteinase-1 is not affected by CK2.
Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I
Authors: EVANS Gareth J.O. , MORGAN Alan
Published: July 2002

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.
Structure–function of the multifunctional Ca2+/calmodulin-dependent protein kinase II
Authors: HUDMON Andy , SCHULMAN Howard
Published: July 2002

Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a ‘molecular switch-like’ behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure—function of this multifunctional protein kinase.
Insulin regulation of hepatic insulin-like growth factor-binding protein-1 (IGFBP-1) gene expression and mammalian target of rapamycin (mTOR) signalling is impaired by the presence of hydrogen peroxide
Authors: PATEL Satish , VAN DER KAAY Jeroen , SUTHERLAND Calum
Published: July 2002

Hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) is rapidly and completely inhibited by insulin. The signalling pathway that mediates this effect of insulin requires the activation of phosphoinositide 3-kinase (PI 3-kinase). Many of the cellular actions of insulin, including activation of PI 3-kinase, can be ‘mimicked’ by oxidative stresses, such as H2O2. In the present study, we demonstrate that H2O2 does not ‘mimic’ but rather antagonizes insulin repression of IGFBP-1 gene expression in H4IIE cells. This effect is accompanied by a decrease in the insulin-induced activation of mammalian target of rapamycin (mTOR)-dependent signalling. However, insulin-induced phosphorylation and regulation of protein kinase B, glycogen synthase kinase-3 and FKHR (forkhead in rhabdomyosarcoma) are not affected by H2O2 in the same cells. In addition, H2O2 strongly activates the p42/p44 mitogen-activated protein kinases, but the presence of PD184352 (an inhibitor of this pathway) does not block the effect of H2O2 on IGFBP-1 gene expression. Our results support the view that the insulin-mediated repression of IGFBP-1 gene expression is partly mTOR-dependent, and demonstrate that H2O2 selectively antagonizes mTOR-dependent insulin action. The implications for the use of H2O2-generating agents as therapeutics for the treatment of insulin resistance, as well as the role of oxidative stress in the development of insulin resistance, are discussed.
Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates
Authors: ATTUCCI Sylvie , KORKMAZ Brice , JULIANO Luiz , HAZOUARD Eric , GIRARDIN Catherine , BRILLARD-BOURDET Michèle , RÉHAULT Sophie , ANTHONIOZ Philippe , GAUTHIER Francis
Published: July 2002

Activated human polymorphonuclear neutrophils at inflammatory sites release the chymotrypsin-like protease cathepsin G, together with elastase and proteinase 3 (myeloblastin), from their azurophil granules. The low activity of cathepsin G on synthetic substrates seriously impairs studies designed to clarify its role in tissue inflammation. We have solved this problem by producing new peptide substrates with intramolecularly quenched fluorescence. These substrates were deduced from the sequence of putative protein targets of cathepsin G, including the reactive loop sequence of serpin inhibitors and the N-terminal domain of the protease-activated receptor of thrombin, PAR-1. Two substrates were selected, Abz-TPFSGQ-EDDnp and Abz-EPFWEDQ-EDDnp, that are cleaved very efficiently by cathepsin G but not by neutrophil elastase or proteinase 3, with specificity constants (kcat/Km) in the 105M-1·s-1 range. They can be used to measure subnanomolar concentrations of free enzyme in vitro and at the surface of neutrophils purified from fresh human blood. Purified neutrophils express 0.02—0.7pg of cathepsin G/cell (n = 15) at their surface. This means that about 104 purified cells may be enough to record cathepsin G activity within minutes. This may be most important for investigating the role of cathepsin G as an inflammatory agent, especially in bronchoalveolar lavage fluids from patients with pulmonary inflammatory disorders.
GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADP-ribosylation factors
Authors: TAKATSU Hiroyuki , YOSHINO Kaori , TODA Kyoko , NAKAYAMA Kazuhisa
Published: July 2002

ADP-ribosylation factors (ARFs) are a family of small GTPases that are involved in various aspects of membrane trafficking events. These include ARF1—ARF6, which are divided into three classes on the basis of similarity in the primary structure: Class I, ARF1—ARF3; Class II, ARF4 and ARF5; and Class III, ARF6. Previous studies identified a novel family of potential ARF effectors, termed GGA1—GGA3, which interact specifically with GTP-bound ARF1 and ARF3 and are localized to the trans-Golgi network (TGN) or its related compartment(s) (GGA is an abbreviation for Golgi-localizing, γ-adaptin ear homology domain, ARF-binding protein). In the present study we have shown that ARF proteins belonging to the three classes, ARF1, ARF5 and ARF6, can interact with all GGA proteins in a yeast two-hybrid assay, in vitro and in vivo. Segmentation of GGA proteins and isolation of GGA mutants defective in ARF binding have revealed that a limited region within the GGA homology domain, which is conserved in the GGA family, is essential for ARF binding. Expression in cells of GTPase-restricted mutants of ARF1 and ARF5 blocks dissociation of GGA proteins from membranes induced by brefeldin A. However, neither of the ARF mutants recruits GGA mutants defective in ARF binding. On the basis of these observations, we conclude that at least ARF1 (Class I) and ARF5 (Class II) in their GTP-bound state cause recruitment of GGA proteins on to TGN membranes. In contrast, on the basis of similar experiments, ARF6 (Class III) may be involved in recruitment of GGA proteins to other compartments, possibly early endosomes.
S–S bonds are not required for the sonochemical formation of proteinaceous microspheres: the case of streptavidin
Authors: AVIVI Sigalit , GEDANKEN Aharon
Published: July 2002

Proteinaceous microspheres can be prepared using the sonochemical method. However, it is known that these proteins should possess at least one cysteine residue in order to obtain stable microspheres using this method. In the present study, we have produced streptavidin microspheres, using the sonochemical method, from streptavidin, which does not have any cysteine residues.
A non-radioactive method for the assay of many serine/threonine-specific protein kinases
Authors: ROSS Heike , ARMSTRONG Christopher G. , COHEN Philip
Published: July 2002

The generation of drugs that modulate the activities of particular protein kinases has become a prime focus of the pharmaceutical and biotechnology industry. Consequently, improved methods for the development of high-throughput screening formats for these enzymes is a high priority. In the present study, we have designed three generic peptide substrates that can be used to assay a diverse range of protein kinases. These peptides share a common seven-residue epitope that includes the site of phosphorylation, and against which we have generated a phospho-specific antibody. Thus a large number of serine/threonine-specific protein kinases can be screened using a simple non-radioactive format.
Fate of haem iron in the malaria parasite Plasmodium falciparum
Authors: EGAN Timothy J. , COMBRINCK Jill M. , EGAN Joanne , HEARNE Giovanni R. , MARQUES Helder M. , NTENTENI Skhumbuzo , SEWELL B. Trevor , SMITH Peter J. , TAYLOR Dale , van SCHALKWYK Donelly A. , WALDEN Jason C.
Published: July 2002

Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61±2% of the iron within parasitized erythrocytes, of which 92±6% is located within the food vacuole. Of this, 88±9% is in the form of haemozoin. 57Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion.