bluuebunny/crossref_metadata_2025 huggingface dataset:
Transketolase from Leishmania mexicana has a dual subcellular localization
Authors: VEITCH Nicola J. , MAUGERI Dante A. , CAZZULO Juan Jose , LINDQVIST Ylva , BARRETT Michael P.
Published: August 2004

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent Km of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 Å (1 Å≡0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.
Superoxide production by NADH:ubiquinone oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner membrane
Authors: LAMBERT Adrian J. , BRAND Martin D.
Published: August 2004

The relationship between protonmotive force and superoxide production by mitochondria is poorly understood. To address this issue, the rate of superoxide production from complex I of rat skeletal muscle mitochondria incubated under a variety of conditions was assessed. By far, the largest rate of superoxide production was from mitochondria respiring on succinate; this rate was almost abolished by rotenone or piericidin, indicating that superoxide production from complex I is large under conditions of reverse electron transport. The high rate of superoxide production by complex I could also be abolished by uncoupler, confirming that superoxide production is sensitive to protonmotive force. It was inhibited by nigericin, suggesting that it is more dependent on the pH gradient across the mitochondrial inner membrane than on the membrane potential. These effects were examined in detail, leading to the conclusions that the effect of protonmotive force was mostly direct, and not indirect through changes in the redox state of the ubiquinone pool, and that the production of superoxide by complex I during reverse electron transport was at least 3-fold more sensitive to the pH gradient than to the membrane potential.
Active-site-mediated elimination of hydrogen fluoride from a fluorinated substrate analogue by isopenicillin N synthase
Authors: GRUMMITT Annaleise R. , RUTLEDGE Peter J. , CLIFTON Ian J. , BALDWIN Jack E.
Published: August 2004

Isopenicillin N synthase (IPNS) is a non-haem iron oxidase that catalyses the formation of bicyclic isopenicillin N from δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV). In this study we report a novel activity for the iron of the IPNS active site, which behaves as a Lewis acid to catalyse the elimination of HF from the fluorinated substrate analogue, δ-(L-α-aminoadipoyl)-L-cysteinyl-D-β-fluorovaline (ACβFV). X-Ray crystallographic studies of IPNS crystals grown anaerobically with ACβFV reveal that the valinyl β-fluorine is missing from the active site region, and suggest the presence of the unsaturated tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-isodehydrovaline in place of substrate ACβFV. 19F NMR studies confirm the release of fluoride from ACβFV in the presence of the active IPNS enzyme. These results suggest a new mode of reactivity for the IPNS iron centre, a mechanism of action that has not previously been reported for any of the iron oxidase enzymes.
Functions of NF-κB1 and NF-κB2 in immune cell biology
Authors: BEINKE Sören , LEY Steven C.
Published: August 2004

Two members of the NF-κB (nuclear factor κB)/Rel transcription factor family, NF-κB1 and NF-κB2, are produced as precursor proteins, NF-κB1 p105 and NF-κB2 p100 respectively. These are proteolytically processed by the proteasome to produce the mature transcription factors NF-κB1 p50 and NF-κB2 p52. p105 and p100 are known to function additionally as IκBs (inhibitors of NF-κB), which retain associated NF-κB subunits in the cytoplasm of unstimulated cells. The present review focuses on the latest advances in research on the function of NF-κB1 and NF-κB2 in immune cells. NF-κB2 p100 processing has recently been shown to be stimulated by a subset of NF-κB inducers, including lymphotoxin-β, B-cell activating factor and CD40 ligand, via a novel signalling pathway. This promotes the nuclear translocation of p52-containing NF-κB dimers, which regulate peripheral lymphoid organogenesis and B-lymphocyte differentiation. Increased p100 processing also contributes to the malignant phenotype of certain T- and B-cell lymphomas. NF-κB1 has a distinct function from NF-κB2, and is important in controlling lymphocyte and macrophage function in immune and inflammatory responses. In contrast with p100, p105 is constitutively processed to p50. However, after stimulation with agonists, such as tumour necrosis factor-α and lipopolysaccharide, p105 is completely degraded by the proteasome. This releases associated p50, which translocates into the nucleus to modulate target gene expression. p105 degradation also liberates the p105-associated MAP kinase (mitogen-activated protein kinase) kinase kinase TPL-2 (tumour progression locus-2), which can then activate the ERK (extracellular-signal-regulated kinase)/MAP kinase cascade. Thus, in addition to its role in NF-κB activation, p105 functions as a regulator of MAP kinase signalling.
Two conserved cysteine residues are critical for the enzymic function of the human platelet-derived growth factor receptor-β: evidence for different roles of Cys-822 and Cys-940 in the kinase activity
Authors: LEE Joon-Won , KIM Jee-Eun , PARK Eun-Jung , KIM Jin-Hyun , LEE Chang-Hun , LEE Seung-Rock , KWON Jongbum
Published: August 2004

The platelet-derived growth factor receptor-β (PDGFR-β) has a number of conserved cysteine residues on its cytoplasmic domain. We have examined whether the cysteine residues play a role in the enzymic function of PDGFR-β. We found that N-ethylmaleimide, which selectively alkylates free thiol groups of cysteine residues, completely inhibited the kinase activity of PDGFR-β. We then identified, through site-directed mutagenesis, two conserved cysteine residues critical for the enzymic function of PDGFR-β. Cys to Ser mutations for either Cys-822, positioned in the catalytic loop, or Cys-940, located in the C-terminal kinase subdomain, significantly reduced the activities of autophosphorylation and phosphorylation towards exogenous substrates. The non-reducing gel analysis indicated that neither of these cysteine residues contributes to the kinase activity by disulphide-bond formation. In addition, the individual mutation of Cys-822 and Cys-940 had no effect on protein stability or the binding of substrates or ATP, implying that these cysteine residues are involved in enzyme catalysis. Finally, proteolytic cleavage assays showed that the mutation of Cys-940, but not Cys-822, induced a protein conformational change. Taken together, these results suggest that Cys-940 contributes to the catalytic activity of PDGFR-β by playing a structural role, whereas Cys-822 contributes through a different mechanism.
Kinetics and control of oxidative phosphorylation in rat liver mitochondria after dexamethasone treatment
Authors: ROUSSEL Damien , DUMAS Jean-François , SIMARD Gilles , MALTHIÈRY Yves , RITZ Patrick
Published: August 2004

The present investigation was undertaken in order to evaluate the contributions of ATP synthesis and proton leak reactions to the rate of active respiration of liver mitochondria, which is altered following dexamethasone treatment (1.5 mg/kg per day for 5 days). We applied top-down metabolic control analysis and its extension, elasticity analysis, to gain insight into the mechanisms of glucocorticoid regulation of mitochondrial bioenergetics. Liver mitochondria were isolated from dexamethasone-treated, pair-fed and control rats when in a fed or overnight fasted state. Injection of dexamethasone for 5 days resulted in an increase in the fraction of the proton cycle of phosphorylating liver mitochondria, which was associated with a decrease in the efficiency of the mitochondrial oxidative phosphorylation process in liver. This increase in proton leak activity occurred with little change in the mitochondrial membrane potential, despite a significant decrease in the rate of oxidative phosphorylation. Regulation analysis indicates that mitochondrial membrane potential homoeostasis is achieved by equal inhibition of the mitochondrial substrate oxidation and phosphorylation reactions in rats given dexamethasone. Our results also suggest that active liver mitochondria from dexamethasone-treated rats are capable of maintaining phosphorylation flux for cellular purposes, despite an increase in the energetic cost of mitochondrial ATP production due to increased basal proton permeability of the inner membrane. They also provide a complete description of the effects of dexamethasone treatment on liver mitochondrial bioenergetics.
Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase
Authors: WINAYANUWATTIKUN Pakorn , KETTERMAN Albert J.
Published: August 2004

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.
Electrostatic interactions of domain III stabilize the inactive conformation of μ-calpain
Authors: FERNÁNDEZ-MONTALVÁN Amaury , ASSFALG-MACHLEIDT Irmgard , PFEILER Dietmar , FRITZ Hans , JOCHUM Marianne , MACHLEIDT Werner
Published: August 2004

The ubiquitous μ- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human μ-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive μ-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.
Platelet cell-surface protein disulphide-isomerase mediated S-nitrosoglutathione consumption
Authors: ROOT Paul , SLISKOVIC Inga , MUTUS Bulent
Published: August 2004

S-nitrosothiols (RSNOs) regulate several aspects of platelet physiology including inhibition of activation, adhesion and aggregation. PDI (protein disulphide-isomerase) has recently been found to be localized to the cell surface, where it exhibits both disulphide-exchange and denitrosation activities. The disulphide-exchange activity of PDI has been linked to aspects of platelet aggregation. The present study suggests that the metabolism of RSNOs by platelets is a function of PDI denitrosation activity. Exposure of washed human platelets to increasing concentrations of GSNO (S-nitrosoglutathione) resulted in saturable denitrosation kinetics. The presence of known PDI inhibitors phenylarsine oxide and anti-PDI antibodies prevented GSNO denitrosation. The fact that, in the presence of GSNO plus the cell-permeable guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one, the initial rates of ADP-induced platelet aggregation and the maximum ΔOD were diminished by ∼40% shows that RSNOs have dual inhibitory effects on platelets, which are mediated through PDI. First, PDI denitrosates RSNOs, releasing NO that, via the guanylate cyclase/G-kinase route, attenuates platelet activation. Secondly, RSNOs are denitrosated at the same PDI-active site that catalyses the disulphide bond formation between integrins and their ligands, thereby attenuating irreversible aggregation.
Activation of caspases-8 and -10 by FLIPL
Authors: BOATRIGHT Kelly M. , DEIS Cristina , DENAULT Jean-Bernard , SUTHERLIN Daniel P. , SALVESEN Guy S.
Published: August 2004

The first step in caspase activation is transition of the latent zymogen to an active form. For the initiator caspases, this occurs through dimerization of monomeric zymogens at an activating complex. Recent studies have suggested that FLIPL [FLICE-like inhibitory protein, long form; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1β-converting enzyme], previously thought to act solely as an inhibitor of caspase-8 activation, can under certain circumstances function to enhance caspase activation. Using an in vitro induced-proximity assay, we demonstrate that activation of caspases-8 and -10 occurs independently of cleavage of either the caspase or FLIPL. FLIPL activates caspase-8 by forming heterodimeric enzyme molecules with substrate specificity and catalytic activity indistinguishable from those of caspase-8 homodimers. Significantly, the barrier for heterodimer formation is lower than that for homodimer formation, suggesting that FLIPL is a more potent activator of caspase-8 than is caspase-8 itself.
p90 ribosomal S6 kinase 2 is associated with and dephosphorylated by protein phosphatase 2Cδ
Authors: DOEHN Ulrik , GAMMELTOFT Steen , SHEN Shi-Hsiang , JENSEN Claus J.
Published: August 2004

RSK2 (p90 ribosomal S6 kinase 2) is activated via the ERK (extracellular-signal-regulated kinase) pathway by phosphorylation on four sites: Ser227 in the activation loop of the N-terminal kinase domain, Ser369 in the linker, Ser386 in the hydrophobic motif and Thr577 in the C-terminal kinase domain of RSK2. In the present study, we demonstrate that RSK2 is associated in vivo with PP2Cδ (protein phosphatase 2Cδ). In epidermal growth factorstimulated cells, RSK2 is partially dephosphorylated on all four sites in an Mn2+-dependent manner, leading to reduced protein kinase activity. Furthermore, PP2Cδ is phosphorylated by ERK on Thr315 and Thr333 in the catalytic domain. Mutation of Thr315 and Thr333 to alanine in a catalytically inactive mutant PP2Cδ(H154D) (His154→Asp) increases the association with RSK2 significantly, whereas mutation to glutamate, mimicking phosphorylation, reduces the binding of RSK2. The domains of interaction are mapped to the N-terminal extension comprising residues 1–71 of PP2Cδ and the N-terminal kinase domain of RSK2. The interaction is specific, since PP2Cδ associates with RSK1–RSK4, MSK1 (mitogen- and stress-activated kinase 1) and MSK2, but not with p70 S6 kinase or phosphoinositide-dependent kinase 1. We conclude that RSK2 is associated with PP2Cδ in vivo and is partially dephosphorylated by it, leading to reduced kinase activity.
The flip side of FLIP
Authors: PETER Marcus E.
Published: August 2004

Two major pathways regulate apoptosis induction in mammalian cells. In the extrinsic pathway, apoptosis is induced through specialized surface receptors, whereas in the intrinsic pathway, apoptosis is induced from within the cell, mainly through activation of mitochondria. Shortly after the major mediators of the extrinsic apoptosis pathway, the initiator caspases-8 and -10, were identified, c-FLIP [FLICE-like inhibitory protein; FLICE is FADD (Fas-associated death domain protein)-like interleukin-1β-converting enzyme], a catalytically inactive caspase-8/-10 homologue, was reported. Whether this structure acts as an inhibitor or promoter of the extrinsic apoptosis pathway has been the subject of much debate. In this issue of the Biochemical Journal, Boatright et al. provide further evidence for the long splice form of c-FLIP (c-FLIPL) being an activator of caspase-8/-10, and demonstrate that the resulting heterodimer is enzymically active with a substrate specificity identical with that of the caspase-8 homodimer. Our understanding of the regulators of the extrinsic apoptosis signalling pathway biochemically may provide the means to design drugs to correct the imbalance between apoptosis and proliferation, as found in many diseases.
An integrated evaluation of endothelial constitutive nitric oxide synthase polymorphisms and coronary artery disease in men
Authors: AGEMA Willem R. P. , de MAAT Moniek P. M.  , ZWINDERMAN Aeilko H. , KASTELEIN John J. P. , RABELINK Ton J. , van BOVEN Ad J. , FESKENS Edith J. M. , BOER Jolanda M. A. , van der WALL Ernst E. , JUKEMA J. Wouter
Published: August 2004

In the present study, we sought to evaluate the role of three polymorphisms in the ecNOS (endothelial constitutive nitric oxide synthase) gene in relation to the existence, severity and progression of CAD (coronary artery disease), MI (myocardial infarction) and the occurrence of ischaemia in a predominantly Caucasian population. Patients with CAD (n=760) and age- and sex-matched population-based controls (n=691) were genotyped for the −786T/C, E/D298 and 4a/b polymorphisms. Patients were randomized to pravastatin (40 mg) or placebo. Progression of atherosclerosis was evaluated by sequential angiography. Functionality was assessed by ST segment analysis of ambulant ECGs. The E298 (P=0.003) and 4a (P=0.001) alleles were associated with CAD. Furthermore, E298 (P=0.009) and −786T (P=0.022) alleles were associated with previous MI among patients, predominantly smokers. D/D298 homozygotes, but not −786T/C or 4a/4b mutants, had longer-lasting ischaemia than others (P<0.05). We found no differences in progression of atherosclerosis, irrespective of pravastatin use. We conclude that the E/D298 polymorphism is most consistently associated with CAD, but not with progression of atherosclerosis. The E allele is associated with CAD and MI, whereas the D allele is associated with ischaemia.
Oral glutamine attenuates indomethacin-induced small intestinal damage
Authors: BASIVIREDDY Jayasree , JACOB Molly , BALASUBRAMANIAN Kunissery A.
Published: August 2004

The use of NSAIDs (non-steroidal anti-inflammatory drugs), although of great therapeutic value clinically, is limited by their tendency to cause mucosal damage in the gastrointestinal tract. In the small intestine, the effects these drugs have been shown to produce include inhibition of cyclo-oxygenase, mitochondrial dysfunction and free radical-induced oxidative changes, all of which contribute to the mucosal damage seen. Glutamine is a fuel preferentially used by enterocytes and is known to contribute to maintaining the integrity of these cells. In the present study, we investigated the effect of glutamine on indomethacin-induced changes in the small intestinal mucosa. Rats were given 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine or alanine, in the diet for 7 days. Indomethacin was then administered orally at a dose of 40 mg/kg of body weight. After 1 h, the small intestine was removed and used for the measurement of parameters of oxidative stress and mitochondrial and BBM (brush border membrane) function. Evidence of oxidative stress was found in the mucosa of the small intestine of drug-treated rats, as indicated by significantly increased activity of xanthine oxidase (P<0.001) and myeloperoxidase (P<0.001), with corresponding decreases in the levels of several free radical scavenging enzymes and α-tocopherol (P<0.001 in all cases). Levels of products of peroxidation were also significantly elevated (P<0.001 for all the parameters measured). In addition, oxidative stress was evident in isolated intestinal mitochondria and BBMs (P<0.001 for all the parameters measured), with associated alterations in function of these organelles (P<0.001 for all the parameters measured). Supplementation of the diet with glutamine or glutamic acid prior to treatment with indomethacin produced significant amelioration in all the effects produced by the drug in the small intestine (P<0.001 for all the parameters measured). Glycine and alanine were found to be much less effective in these respects.
Plasma expansion by polyethylene-glycol-modified albumin
Authors: ASSALY Ragheb A. , AZIZI Mustafa , KENNEDY David J. , AMAURO Cristine , ZAHER Aiman , HOUTS Frederick W. , HABIB Robert H. , SHAPIRO Joseph I. , DIGNAM J. David
Published: August 2004

Systemic inflammatory response conditions are associated with capillary leak and haemodynamic compromise. Fluid resuscitation to reverse the ensuing hypovolaemia is, however, complicated by the decreased endothelium reflection coefficient to albumin and other colloids. We developed PEG–Alb (albumin covalently linked to polyethylene glycol) as a potential resuscitative agent. PEG was covalently linked to human albumin at multiple sites on the protein. The modified protein was heterogeneous when examined by SDS/PAGE, size-exclusion chromatography and SELDI–TOF MS (surface-enhanced laser-desorption ionization–time of flight MS). Based on size-exclusion chromatography and osmotic pressure data, the effective volume of PEG–Alb is increased 13- to 16-fold compared with unmodified albumin. In an LPS (lipopolysaccharide) model of shock, rats treated with PEG–Alb showed better blood pressure, lower Hct (haematocrit) consistent with haemodilution and less lung injury than rats treated with unmodified albumin or saline. In a CLP (caecal ligation and puncture) model of sepsis, PEG–Alb was more effective than albumin or saline in maintaining blood pressure and in decreasing Hct. When fluorescein-labelled PEG–Alb and Texas Red-labelled albumin were administered to rats with LPS- or CLP-induced shock, PEG–Alb was retained within blood vessels, whereas albumin extravasates into the interstitial space. Based on these data, PEG–Alb appears to be retained within blood vessels in models of capillary leak. PEG–Alb may ultimately be effective in the clinical treatment of shock associated with capillary leak.
Effect of esmolol on positive-pressure ventilation-induced variations of arterial pressure in anaesthetized humans
Authors: LAI Hsien Yong , YANG Cheryl C. H. , CHENG Ching-Feng , HUANG Fan Yen , LEE Yi , SHYR Ming-Hwang , KUO Terry B. J.
Published: August 2004

Positive-pressure ventilation-induced variations in arterial pressure have been related to cardiac sympathetic activity in animals. However, the effect of β-adrenoceptor blockade on these variations in anaesthetized humans under positive-pressure ventilation has not yet been investigated. In the present study, RAPV (respiratory-related arterial pressure variability) and %SPV (percentile systolic pressure variation) were determined before and after esmolol treatment in ten mechanically ventilated patients. RAPV and %SPV decreased significantly after intravenous esmolol (1 mg/kg of body weight) treatment (maximal decrease of RAPV, 50% and %SPV, 35%). Linear regression analysis of RAPV and %SPV before and after esmolol treatment both revealed high correlation (r=0.93 and 0.91 respectively). The amplitudes of RAPV and %SPV also significantly increased in a graded way with higher tidal volumes. Thus we propose that esmolol suppresses the variations in arterial pressure induced by positive-pressure mechanical ventilation, and we suggest that RAPV and %SPV may be alternative choices for monitoring cardiac sympathetic regulation in anaesthetized patients under positive-pressure ventilation.
Short-term use of continuous positive airway pressure ameliorates glomerular hyperfiltration in patients with obstructive sleep apnoea syndrome
Authors: KINEBUCHI Shin-ichi , KAZAMA Junichiro J. , SATOH Makoto , SAKAI Kunihiko , NAKAYAMA Hideaki , YOSHIZAWA Hirohisa , NARITA Ichiei , SUZUKI Eiichi , GEJYO Fumitake
Published: August 2004

Patients with OSAS (obstructive sleep apnoea syndrome) demonstrate renal signs such as proteinuria, glomerular hypertrophy and focal glomerular sclerosis. We performed a clinical study to investigate the glomerular function in OSAS patients and the short-term effect of CPAP (continuous positive airway pressure) on it. OSAS patients underwent a sodium thiosulphate and p-aminohippurate double clearance test, polysomnography and ambulatory blood pressure monitoring before and a week after the induction of CPAP. Twenty-seven consecutive patients (24 males) with moderate-to-severe OSAS admitted to our hospital for the induction of CPAP, and 32 healthy donors for renal transplantation as controls participated in the study. Before treatment, the glomerular filtration rate, estimated by the sodium thiosulphate clearance test, was within normal range, and the renal plasma flow was significantly lower than normal in the OSAS patients, thus the FF (filtration fraction) value was much higher than normal. FF before CPAP was not significantly correlated with age, body mass index or blood pressure; however, indices of increased hypoxaemia correlated with increased FF values. Polysomnographic variables after CPAP showed significant improvements in all patients, and only the nocturnal blood pressures were slightly lower than before CPAP. In 21 patients who underwent the clearance test after CPAP, FF significantly decreased from 0.26±0.04 to 0.23±0.03 (P<0.001). OSAS patients were generally in a glomerular-hyperfiltrating condition that appeared to cause the renal findings associated with OSAS. CPAP might prevent nephropathy by ameliorating the glomerular hyperfiltration in OSAS patients.
Effects of pro- and anti-inflammatory cytokines and nitric oxide donors on hyaluronic acid synthesis by synovial cells from patients with rheumatoid arthritis
Authors: CHENEVIER-GOBEAUX Camille , MORIN-ROBINET Séverine , LEMARECHAL Hervé , POIRAUDEAU Serge , EKINDJIAN Jean-Charles O. G. , BORDERIE Didier
Published: August 2004

The aim of the present study was to investigate the effects of (i) the pro-inflammatory cytokines IL (interleukin)-1β, TNF-α (tumour necrosis factor-α), IFN-γ (interferon-γ) and anti-inflammatory cytokines IL-4 and IL-13, and (ii) NO (nitric oxide) donors on HA (hyaluronic acid) production by synovial cells from patients with rheumatoid arthritis. Synovial cells obtained from five patients with rheumatoid arthritis were incubated for 24 h without or with IL-1β, TNF-α, IFN-γ, or with this mixture for 24 h plus IL-4 or IL-13 for the last 6 h. The same cells were also incubated for 3–24 h without or with SNP (sodium nitroprusside) or SNAP (S-nitroso-N-acetyl-DL-penicillamine). HA secretion was determined by an immunoenzymic assay based on HA-specific binding by proteoglycan isolated from bovine cartilage. IL-1β, TNF-α and IFN-γ alone or in combination stimulated HA synthesis, whereas IL-4 and IL-13 dose-dependently inhibited HA production induced by Th1 cytokines. HA production was significantly increased by the presence of 1 mM SNP after 6 and 12 h (maximal effect). HA production was significantly increased by the presence of 0.01 and 0.1 mM SNAP after 12 h of incubation, and cells treated with 1 mM SNAP showed a maximal HA production after 24 h of incubation. In conclusion, the present study provides data concerning the regulatory role of pro- and anti-inflammatory cytokines and NO donors on HA metabolism in rheumatoid synovial cells and may help in understanding the pathophysiology of rheumatoid arthritis.
Lipoprotein transport in the metabolic syndrome: methodological aspects of stable isotope kinetic studies
Authors: CHAN Dick C. , BARRETT P. Hugh R. , WATTS Gerald F.
Published: August 2004

The metabolic syndrome encapsulates visceral obesity, insulin resistance, diabetes, hypertension and dyslipidaemia. Dyslipidaemia is a cardinal feature of the metabolic syndrome that accelerates the risk of cardiovascular disease. It is usually characterized by high plasma concentrations of triacylglycerol (triglyceride)-rich and apoB (apolipoprotein B)-containing lipoproteins, with depressed concentrations of HDL (high-density lipoprotein). However, lipoprotein metabolism is complex and abnormal plasma concentrations can result from alterations in the rates of production and/or catabolism of these lipoprotein particles. Our in vivo understanding of kinetic defects in lipoprotein metabolism in the metabolic syndrome has been achieved chiefly by ongoing developments in the use of stable isotope tracers and mathematical modelling. This review deals with the methodological aspects of stable isotope kinetic studies. The design of in vivo turnover studies requires considerations related to stable isotope tracer administration, duration of sampling protocol and interpretation of tracer data, all of which are critically dependent on the kinetic properties of the lipoproteins under investigation. Such models provide novel insight that further understanding of metabolic disorders and effects of treatments. Future investigations of the pathophysiology and therapy of the dyslipoproteinaemia of the metabolic syndrome will require the development of novel kinetic methodologies. Specifically, new stable isotope techniques are required for investigating in vivo the turnover of the HDL subpopulation of particles, as well as the cellular efflux of cholesterol into the extracellular space and its subsequent transport in plasma and metabolic fate in the liver.
Lipoprotein transport in the metabolic syndrome: pathophysiological and interventional studies employing stable isotopy and modelling methods
Authors: CHAN Dick C. , BARRETT P. Hugh R. , WATTS Gerald F.
Published: August 2004

The accompanying review in this issue of Clinical Science [Chan, Barrett and Watts (2004) Clin. Sci. 107, 221–232] presented an overview of lipoprotein physiology and the methodologies for stable isotope kinetic studies. The present review focuses on our understanding of the dysregulation and therapeutic regulation of lipoprotein transport in the metabolic syndrome based on the application of stable isotope and modelling methods. Dysregulation of lipoprotein metabolism in metabolic syndrome may be due to a combination of overproduction of VLDL [very-LDL (low-density lipoprotein)]-apo (apolipoprotein) B-100, decreased catabolism of apoB-containing particles and increased catabolism of HDL (high-density lipoprotein)-apoA-I particles. These abnormalities may be consequent on a global metabolic effect of insulin resistance, partly mediated by depressed plasma adiponectin levels, that collectively increases the flux of fatty acids from adipose tissue to the liver, the accumulation of fat in the liver and skeletal muscle, the hepatic secretion of VLDL-triacylglycerols and the remodelling of both LDL (low-density lipoprotein) and HDL particles in the circulation. These lipoprotein defects are also related to perturbations in both lipolytic enzymes and lipid transfer proteins. Our knowledge of the pathophysiology of lipoprotein metabolism in the metabolic syndrome is well complemented by extensive cell biological data. Nutritional modifications may favourably alter lipoprotein transport in the metabolic syndrome by collectively decreasing the hepatic secretion of VLDL-apoB and the catabolism of HDL-apoA-I, as well as by potentially increasing the clearance of LDL-apoB. Several pharmacological treatments, such as statins, fibrates or fish oils, can also correct the dyslipidaemia by diverse kinetic mechanisms of action, including decreased secretion and increased catabolism of apoB, as well as increased secretion and decreased catabolism of apoA-I. The complementary mechanisms of action of lifestyle and drug therapies support the use of combination regimens in treating dyslipoproteinaemia in subjects with the metabolic syndrome.
Resolving inconsistency in the results of genetic association studies of cardiovascular disease
Authors: HINGORANI Aroon
Published: August 2004

In this issue of Clinical Science, Agema and co-workers report the results of a genetic association study of eNOS (endothelial nitric oxide synthase) gene polymorphisms (−786T→C, intron 4b→a and Glu298→Asp) in patients with angiographic CAD (coronary artery disease), and/or prior MI (myocardial infarction) and a group of healthy population-based controls. However, the findings of this study appear to contradict previous studies published on the eNOS polymorphisms, and this commentary will attempt to resolve the inconsistency in such genetic association studies.
Neuburgia novocaledonica, comb. nov. and the first record of domatia in the family Loganiaceae
Authors: Molina Jeanmaire E. , Struwe Lena
Published: August 2004

The New Caledonian species Couthovia novocaledonica Gilg & Benedict (Loganiceae) is supported as a species distinct from Neuburgia corynocarpa (A.Gray) Leenh. Neuburgia novocaledonica is different in having domatia on its abaxial leaf surfaces, two rings of hairs within the corolla tube, and generally obovate leaf shape. Therefore, the new combination Neuburgia novocaledonica (Gilg & Benedict) J. Molina & Struwe is hereby made. The presence and morphology of domatia on the lower surface of the leaves of Neuburgia novocaledonica are discussed. This appears to be the first record of domatium occurrence in Loganiaceae.
Evaluation of two relative-abundance indices to monitor brushtail possums in New Zealand
Authors: Warburton Bruce , Barker Richard , Coleman Morgan
Published: August 2004

Population monitoring of brushtail possums (Trichosurus vulpecula) is an essential part of their management in New Zealand, with a trap-catch removal method being used most commonly. An alternative monitoring method (bait-interference), using bite marks on wax blocks, has been promoted as a more cost-effective alternative to using traps. However, neither of these methods has been validated. We assessed the utility of these two methods regarding their accuracy (unbiasedness and precision) in detecting changes in possum abundance by comparing the estimates of relative change in possum density following control obtained from both methods with the kill rate among radio-collared possums in the same study areas. In each of seven control operations, 48–50 possums were collared with mortality-sensing radio-transmitters, and trap-catch and wax-block lines were assessed before and after control. The correlation between trap-catch and radio-transmitter kill estimates (R2 = 0.91) suggests that trap catch, as currently used to monitor relative possum abundance, appears to be sufficiently accurate to manage these pests, with any bias being small. The kill estimates based on the wax-block monitoring were correlated less strongly with the radiotransmitter estimates than the trap-catch estimates (R2 = 0.66), although still significant. Until the extent of the potential bias in the wax-block estimates is known, we recommend that traps continue to be used as the main method to monitor possum abundance.
Managing confined populations: the influence of density on the home range and habitat use of reintroduced burrowing bettongs (Bettongia lesueur)
Authors: Finlayson Graeme R. , Moseby Katherine E.
Published: August 2004

The home range, reproductive condition and warren and habitat use of reintroduced female burrowing bettongs was compared within two enclosures in the Arid Recovery Reserve at estimated densities of 2.75 and 7.5 bettongs km–2. Bettongs at both densities exhibited similar behaviour, with females using an average of 2.7 warrens over six months and home ranges averaging 29 and 35 ha in the low- and high-density enclosure respectively. All five female bettongs studied in the low-density enclosure were carrying pouch young at the beginning and end of the six-month study but only one of the five females in the high-density enclosure was carrying pouch young after six months. Higher food availability may have accounted for the higher reproductive effort and slightly smaller home ranges observed in the low-density enclosure. Female bettongs at both densities favoured dune habitat over chenopod swales and all burrows were in dune habitat. Although some differences in reproductive output were observed, it is likely that densities are not yet high enough to cause significant behavioural changes.
Automated feeders: new technology for food supplementation experiments with mammals
Authors: Isaac Joanne L. , Johnson Christopher N. , Grabau Peter J. , Krockenberger Andrew K.
Published: August 2004

Experimental manipulations of food supply in wild populations are often confounded by a number of factors, such as induced immigration to local populations receiving food supplements and intraspecific competition for access to feed stations. Here, we describe newly developed automated feeding stations, designed to dispense weighed food supplements to specific experimental animals. The feeders incorporate a data logger that records the identification and weight of each animal and the time of the visit to the feeder.Using the common brushtail possum (Trichosurus vulpecula) as a model medium-sized mammal, we present results of an 18-month field trial. Each free-ranging experimental possum was fitted with a collar containing a transponder chip, allowing them access to a feeder. During the field trial, experimental possums were found to show a significant increase in body mass compared with control animals, which showed a slight decrease in mass. Body masses recorded by the feeders for experimental possums did not differ from mass data recorded during live-trapping sessions.The automated feeding stations represent an advance over previous methodology and the first time supplementary food has been delivered in a controlled automated fashion in a wild mammal population. The implications of the feeders to future studies of resource supplementation in mammals are discussed.
Murine cytomegalovirus (MCMV) infections in house mice: a matter of age or sex?
Authors: Jacob Jens , Sutherland Duncan R.
Published: August 2004

The murine cytomegalovirus (MCMV) may be used as a vector for fertility control in house mice, which are a significant agricultural pest in south-eastern Australia. The transmission of MCMV can be density dependent and is mostly subclinical in its effect on mice; however, little is known about which individual parameters influence whether an animal is likely to be infected or not.We examined the impact of age and reproductive history on seroprevalence of MCMV in house mice during the breeding season. MCMV seroprevalence was density dependent and there was a linear increase in seroprevalence with increasing age, starting at 25% for 1-month-old mice and 100% for all individuals older than five months. There was no clear indication that the number of previous pregnancies was related to MCMV seroprevalence.The high prevalence of MCMV among adult female house mice (84%) implies that an Australian field strain of MCMV could be a promising vector for an immunocontraceptive for house mice.
Do dietary changes of Australasian gannets (Morus serrator) reflect variability in pelagic fish stocks?
Authors: Bunce Ashley
Published: August 2004

The relationship between changes in the diet of Australasian gannets (Morus serrator) and commercial fisheries landings was investigated. The contribution of pilchard (Sardinops sagax) to the diet of gannets was significantly related to the commercial catch of that species, suggesting that changes in the relative proportion of pilchard in the diet of gannets may be a useful index of pilchard abundance and availability. Equivalent relationships, however, were not found for other common prey species in the gannet diet. It is considered that the abundance of important prey items in the gannet diet, such as pilchards, and commercial landings are proportional to stock abundance. The implications of this relationship for fisheries management are examined.
Multiple pathways process stalled replication forks
Authors: Michel Bénédicte , Grompone Gianfranco , Florès Maria-Jose , Bidnenko Vladimir
Published: August 2004

Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability. Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery. Several strategies for the processing of inactivated replication forks before replisome reassembly have been described. Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest. The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage. These various restart pathways may reflect different structures at stalled forks. We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes.
Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide
Authors: Lillig Christopher Horst , Lönn Maria Elisabet , Enoksson Mari , Fernandes Aristi Potamitou , Holmgren Arne
Published: August 2004

Glutaredoxin (Grx) belongs to the thioredoxin fold superfamily and catalyzes glutathione-dependent oxidoreductions. The recently discovered mitochondrial and nuclear Grx (Grx2) differs from the more abundant cytosolic Grx (Grx1) by its higher affinity toward
S
-glutathionylated proteins and by being a substrate for thioredoxin reductase. Here, we have successfully established a method to silence the expression of Grx2 in HeLa cells by using short interfering RNA to study its role in the cell. Cells with levels of Grx2 <3% of the control were dramatically sensitized to cell death induced by doxorubicin/adriamycin and phenylarsine oxide but did not show signs of a general increase in oxidative damage with respect to carbonylation and glutathionylation. The ED
50
for doxorubicin dropped from 40 to 0.7 μM and for phenylarsine oxide from 200 to 5 nM. However, no differences were detected after treatment with cadmium, a known inhibitor of Grx1. These results indicate a crucial role of Grx2 in the regulation of the mitochondrial redox status and regulation of cell death at the mitochondrial checkpoint.
Deletion of the Ca2+-activated potassium (BK) α-subunit but not the BKβ1-subunit leads to progressive hearing loss
Authors: Rüttiger Lukas , Sausbier Matthias , Zimmermann Ulrike , Winter Harald , Braig Claudia , Engel Jutta , Knirsch Martina , Arntz Claudia , Langer Patricia , Hirt Bernhard , Müller Marcus , Köpschall Iris , Pfister Markus , Münkner Stefan , Rohbock Karin , Pfaff Imke , Rüsch Alfons , Ruth Peter , Knipper Marlies
Published: August 2004

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming α-subunit coexpressed with the auxiliary β1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel α-(BKα–/–) and β1-subunit (BKβ1–/–) knockout mice, we demonstrate normal hearing function and cochlear structure of BKβ1–/–mice. During the first 4 postnatal weeks also, BKα–/–mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKα–/–mice only from ≈8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-codingslo1gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.
Haploinsufficiency for the murine orthologue of
Chlamydomonas
PF20 disrupts spermatogenesis
Authors: Zhang Zhibing , Kostetskii Igor , Moss Stuart B. , Jones Brian H. , Ho Clement , Wang Hongbin , Kishida Tatsuro , Gerton George L. , Radice Glenn L. , Strauss Jerome F.
Published: August 2004

PF20 was first identified in
Chlamydomonas rheinhardtii
as an essential component of the axoneme central apparatus. We discovered that the mouse
Pf20
gene encodes two major transcripts (2.5 and 1.4 kb), which are expressed in different patterns during spermatogenesis, yielding proteins of 71 and 35 kDa, respectively. Both proteins contain contiguous WD repeats in their C termini. The meiotically expressed 71-kDa protein is incorporated into the central apparatus, whereas the 35-kDa protein, which accumulates in postmeiotic male germ cells, is abundant in the nucleus. We disrupted the
Pf20
gene domains that encode the C-terminal WD repeats in embryonic stem cells. Highly chimeric mice carrying the mutant
Pf20
allele had impaired spermatogenesis with a significant loss of germ cells at the round spermatid stage, in association with disorganization of sperm axoneme structure. The mutated
Pf20
allele was never transmitted, indicating that
Pf20
haploinsufficiency caused the defects in spermatogenesis. The 35-kDa PF20 protein was shown to bind to meiosis-expressed gene 1 (MEIG1), a chromosome/chromatin-binding protein initially expressed during meiosis but retained in the germ cell nucleus throughout later stages of spermatogenesis. Our findings reveal an essential role for
Pf20
in mouse spermatogenesis, sustaining postmeiotic germ cell viability. The different patterns of expression of the two PF20 proteins suggest the possibility that the
Pf20
gene has multiple functions during spermatogenesis.