bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of an HSV‐tk transgenic mouse model for study of liver damage
Authors: Zhang Yan , Huang Shu‐Zhen , Wang Shu , Zeng Yi‐Tao
Published: April 2005

The herpes simplex virus thymidine kinase/ganciclovir (HSV‐tk/GCV) system that selectively depletes cells expressing HSV‐tk upon treatment with GCV has provided a valuable tool for developing a new animal model expressing the desired tissue damage. In this paper, an HSV‐tk vector with an albumin promoter/enhancer was constructed. Based on the favourable killing effect on Hep‐G2 cells by the recombinant construct, the HSV‐tk transgenic mouse strains were developed. One strain of the TK transgenic mouse (TK5) was studied intensively. Integration of the target gene was confirmed primarily by PCR. Fluorescence in situ hybridization following G‐banding analysis demonstrated that the insertion site was located at 2F1‐G3. The hepatocyte‐specific transcription and expression of HSV‐tkwas verified by reverse transcription (RT)–PCR as well as by immunohistochemical staining. When two second‐generation mice (TK5‐F1 and TK5‐F2) were injected with GCV, the pathogenic alterations in the liver were readily identified, including the appearance of vaculation in the hepatocytes with inflammatory infiltration in the liver, and diffuse proliferation of hepatocytes. In addition, the blood test demonstrates a significant increase of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin. In conclusion, the transgenic mouse model with hepatocyte‐specific expressed HSV‐tk developed hepatitis with administration of GCV, had morphological and clinical chemical characteristics indicative of hepatocellular disease and should be useful for the the study of inducible liver‐specific diseases.
Analysis of active‐site amino‐acid residues of human serum paraoxonase using competitive substrates
Authors: Yeung David T. , Lenz David E. , Cerasoli Douglas M.
Published: April 2005

Serum paraoxonase (PON1) is a calcium‐dependent six‐fold β‐propeller protein structurally similar to the di‐isopropylfluorophosphatase (DFPase) found in the squid Loligo vulgaris. Human serum paraoxonase (HuPON1) has been shown to hydrolyze an array of substrates even though relatively little is known about its physiological role(s) or its catalytic mechanism. Through site‐directed mutagenesis studies, designed from a DFPase‐like homology model, and from a crystal structure of a hybrid PON1 molecule, amino‐acid residues essential for enzyme function, including H115 and F222, have been identified. It was shown previously that, when H115 is replaced with tryptophan, the resulting enzyme hydrolyzes paraoxon but not phenyl acetate. This study shows that, when present simultaneously, phenyl acetate competitively inhibits paraoxon hydrolysis by H115W. Conversely, when F222 is replaced with tyrosine, mutant F222Y can hydrolyze phenyl acetate but not paraoxon. The presence of DFP, an inhibitor of both arylesterase and paraoxonase activities of wild‐type HuPON1 (mean Ki = 0.48 ± 0.15 mm), has no effect on the ability of F222Y to catalyze the hydrolysis of phenyl acetate, suggesting that the F222Y mutant is unable to bind DFP. Together, the results suggest that, in wild‐type HuPON1, H115 and F222 are important in determining substrate binding and specificity, but are not likely to be directly involved in substrate hydrolysis.
Collagenous Alzheimer amyloid plaque component assembles amyloid fibrils into protease resistant aggregates
Authors: Söderberg Linda , Dahlqvist Camilla , Kakuyama Hiroyoshi , Thyberg Johan , Ito Akira , Winblad Bengt , Näslund Jan , Tjernberg Lars O.
Published: April 2005

Recently, a novel plaque‐associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimer's disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC‐P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimer's disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid β‐peptide (Aβ). Here, we examined the effects of CLAC on Aβ fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Aβ fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Aβ was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Aβ fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T‐binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Aβ fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Aβ fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.
Apoptosis‐inhibiting activities of BIR family proteins in Xenopus egg extracts
Authors: Tsuchiya Yuichi , Murai Shin , Yamashita Shigeru
Published: April 2005

In many animal species including Xenopus, ovulated eggs possess an intrinsic apoptotic execution system. This program is inhibited for a limited time by some maternal apoptosis inhibitors, although their molecular properties remain uncharacterized. Baculovirus IAP repeat (BIR) family proteins contain evolutionarily conserved BIR domains and play important roles in apoptosis suppression, and are therefore good candidates as maternal apoptosis inhibitors. We identified four maternal BIR family proteins in Xenopus eggs and, using the biochemical advantages of egg extracts, examined their physiological functions. These molecules included two survivin‐related proteins, xEIAP/XLX, and a possible ortholog of XIAP named xXIAP. The addition of recombinant xXIAP greatly delayed apoptotic execution, whereas the immunodepletion of endogenous xXIAP significantly accelerated the onset of apoptosis. In contrast, xEIAP/XLX was a poor apoptosis inhibitor, and neither of the survivin orthologs showed anti‐apoptotic activity in our assay. Both xEIAP/XLX and xXIAP were degraded by activated caspases, and also by a novel proteolytic system that required the presence of C‐terminal RING finger domain but was insensitive to proteasome inhibition. Our data suggest that the regulation of endogenous xXIAP concentration is important for the survival of Xenopus eggs.
The RNA recognition motif, a plastic RNA‐binding platform to regulate post‐transcriptional gene expression
Authors: Maris Christophe , Dominguez Cyril , Allain Frédéric H.‐T.
Published: April 2005

The RNA recognition motif (RRM), also known as RNA‐binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM–RNA or RRM–protein), we reviewed the structure–function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM–nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM β‐sheet, the two external β‐strands, the loops, the C‐ and N‐termini, or even a second RRM domain allow high RNA‐binding affinity and specific recognition. Protein–RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM‐containing proteins.
Evidence for interactions between domains of TatA and TatB from mutagenesis of the TatABC subunits of the twin‐arginine translocase
Authors: Barrett Claire M. L. , Robinson Colin
Published: April 2005

The twin‐arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter‐subunit interactions occurring within Tat complexes. We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions. We show that substitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three glycines in the TatA TM span completely blocks Tat translocation activity. The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large‐scale distortions. Near‐complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA/B TM spans. We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination. Finally, we have re‐examined the effects of previously analyzed TatABC mutations under conditions of high translocation activity. Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity.
Endogenous expression and protein kinase A‐dependent phosphorylation of the guanine nucleotide exchange factor Ras‐GRF1 in human embryonic kidney 293 cells
Authors: Norum Jens Henrik , Méthi Trond , Mattingly Raymond R. , Levy Finn Olav
Published: April 2005

We have previously reported the Ras‐dependent activation of the mitogen‐activated protein kinases p44 and p42, also termed extracellular signal‐regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs‐coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi‐ and Gq‐coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras‐GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N‐terminal IQ domain, the mechanism of Ras activation through Gs‐coupled receptors is not fully understood. We report the endogenous expression of Ras‐GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs‐coupled 5‐HT7 receptors induced protein kinase A‐dependent phosphorylation of the endogenous human Ras‐GRF1 on Ser927 and of transfected mouse Ras‐GRF1 on Ser916. Ras‐GRF1 overexpression increased basal and serotonin‐stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1–225, including the Ca2+/calmodulin‐binding IQ domain, from Ras‐GRF1 reduced both basal and serotonin‐stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5‐HT7 receptors increased [Ca2+]i, and the serotonin‐induced ERK1/2 phosphorylation was Ca2+‐dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras‐dependent ERK1/2 activation after 5‐HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.
Secondary transporters of the 2HCT family contain two homologous domains with inverted membrane topology and trans re‐entrant loops
Authors: Lolkema Juke S. , Sobczak Iwona , Slotboom Dirk‐Jan
Published: April 2005

The 2‐hydroxycarboxylate transporter (2HCT) family of secondary transporters belongs to a much larger structural class of secondary transporters termed ST3 which contains about 2000 transporters in 32 families. The transporters of the 2HCT family are among the best studied in the class. Here we detect weak sequence similarity between the N‐ and C‐terminal halves of the proteins using a sensitive method which uses a database containing the N‐ and C‐terminal halves of all the sequences in ST3 and involves blast searches of each sequence in the database against the whole database. Unrelated families of secondary transporters of the same length and composition were used as controls. The sequence similarity involved major parts of the N‐ and C‐terminal halves and not just a small stretch. The membrane topology of the homologous N‐ and C‐terminal domains was deduced from the experimentally determined topology of the members of the 2HCT family. The domains consist of five transmembrane segments each and have opposite orientations in the membrane. The N terminus of the N‐terminal domain is extracellular, while the N terminus of the C‐terminal domain is cytoplasmic. The loops between the fourth and fifth transmembrane segment in each domain are well conserved throughout the class and contain a high fraction of residues with small side chains, Gly, Ala and Ser. Experimental work on the citrate transporter CitS in the 2HCT family indicates that the loops are re‐entrant or pore loops. The re‐entrant loops in the N‐ and C‐terminal domains enter the membrane from opposite sides (trans‐re‐entrant loops). The combination of inverted membrane topology and trans‐re‐entrant loops represents a new fold for secondary transporters and resembles the structure of aquaporins and models proposed for Na+/Ca2+ exchangers.
Guanidinium chloride‐ and urea‐induced unfolding of FprA, a mycobacterium NADPH‐ferredoxin reductase
Authors: Shukla Nidhi , Bhatt Anant Narayan , Aliverti Alessandro , Zanetti Giuliana , Bhakuni Vinod
Published: April 2005

The guanidinium chloride‐ and urea‐induced unfolding of FprA, a mycobacterium NADPH‐ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo‐protein was observed. The stabilization of apo‐FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea‐ and guanidinium chloride‐induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.
A critical role of TRPM channel‐kinase for human magnesium transport
Authors: Schlingmann Karl P. , Gudermann Thomas
Published: April 2005

Hereditary disorders of magnesium homeostasis comprise a heterogenous group of diseases mainly affecting the renal conservation of magnesium. In the past few years, genetic studies in affected individuals disclosed the first molecular components of epithelial magnesium transport: the tight junction protein paracellin‐1 (claudin‐16) was discovered as a key player in paracellular magnesium and calcium reabsorption in the thick ascending limb of Henle's loop and the γ‐subunit was identified as a component of renal Na+–K+‐ATPase critical for transcellular magnesium reabsorption in the distal convoluted tubule. However, the molecular identity of proteins directly involved in cellular magnesium transport remained largely unknown until a series of recent studies highlighted the critical role of two members of the transient receptor potential (TRP) family, for body magnesium homeostasis. TRPM6 and TRPM7 belong to the melastatin‐related TRPM subfamily of TRP channels whose eight members exhibit a significant diversity in domain structure as well as cation selectivity and activation mechanisms. Both proteins share the unique feature of an atypical kinase domain at their C‐terminus for which they have been termed ‘chanzymes’ (channels plus enzymes). Whereas electrophysiological and biochemical analyses identified TRPM7 as an important player in cellular magnesium homeostasis, the critical role of TRPM6 for epithelial magnesium transport emerged from the discovery of loss‐of‐function mutations in patients with a severe form of hereditary hypomagnesaemia called primary hypomagnesaemia with secondary hypocalcaemia or HSH. The aim of this review is to summarize the data emerging from molecular genetic, biochemical and electrophysiological studies on these fascinating two new proteins combining ion channel and enzyme functions/properties.
Age is no barrier to muscle structural, biochemical and angiogenic adaptations to training up to 24 months in female rats
Authors: Rossiter H. B. , Howlett R. A. , Holcombe H. H. , Entin P. L. , Wagner H. E. , Wagner P. D.
Published: April 2005

Ageing is associated with reduced transport and utilization of O2, diminishing exercise tolerance. Reductions may occur in cardiac output (delivery), and skeletal muscle oxidative capacity (utilization). To determine the reversibility of the declines in the muscular determinants of these limitations, skeletal muscle morphological, angiogenic and biochemical responses to acute exercise and endurance training were investigated in female Fischer 344 rats (n= 42; seven groups of six rats) aged 6 (Y) and 24 (O) months compared with resting untrained controls (YC, OC). Treadmill training lasted 8 weeks (10 deg incline, 1 h per day, 5 days per week). Two groups ran at maximum tolerated speeds (YTR, OTR), while an additional Y group (YTM) trained at OTR speed. There was no effect of age on vascular endothelial growth factor gene expression in gastrocnemius muscles after acute exercise. Similarly, age did not impair the effects of training, with increases (P < 0.05; ±s.e.m.) occurring in all of the following: 1 h exercise running speed (YTR 92 ± 4%versus OTR 140 ± 25%); citrate synthase (YTR 37 ± 8%versus OTR 97 ± 33%) and β‐hydroxyacyl‐CoA‐dehydrogenase (YTR 31 ± 7%, versus OTR 72 ± 24%) activities; and capillary‐to‐fibre ratio (YTR 5.2 ± 0.2%versus OTR 8.1 ± 0.2%). However, YTM muscle was unchanged in each measure compared with YC. In conclusion, these muscular responses to training were (1) not reduced by ageing, but (2) dependent on relative and not absolute work rate, since, at the same speed, OTR rats showed greater changes than YTM. Therefore, increases in exercise tolerance and muscle adaptations are not impaired in female rats up to 24 months of age, and require a smaller absolute exercise stimulus (than young) to be manifest.
Navigation in space – the role of the macaque ventral intraparietal area
Authors: Bremmer Frank
Published: April 2005

Goal‐directed self‐motion through space is anything but a trivial task. What we take for granted in everyday life requires the complex interplay of different sensory and motor systems. On the sensory side most importantly a target of interest has to be localized relative to one's own position in space. On the motor side the most critical step in neural processing is to define and perform a movement towards the target as well as the avoidance of obstacles. Furthermore, the multisensory (visual, tactile and auditory) motion signals as induced by one's own movement have to be identified and differentiated from the real motion of visual, tactile or auditory objects in the outside world. In a number of experimental studies performed in recent years we and others have functionally characterized a subregion within monkey posterior parietal cortex (PPC) that appears to be well suited to contribute to such multisensory encoding of spatial and motion information. In this review I will summarize the most important experimental findings on the functional properties of this very region in monkey PPC, i.e. the ventral intraparietal area.
ATP contributes to the generation of network‐driven giant depolarizing potentials in the neonatal rat hippocampus
Authors: Safiulina Victoria F. , Kasyanov Alexander M. , Sokolova Elena , Cherubini Enrico , Giniatullin Rashid
Published: April 2005

In the immature hippocampus, the so‐called ‘giant depolarizing potentials’ (GDPs) are network‐driven synaptic events generated by the synergistic action of glutamate and GABA. Here we tested the hypothesis that ATP, a widely distributed neurotransmitter, directly contributes to the network activity during the first postnatal week. We found that in CA3 pyramidal cells, in the presence of the adenosine antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), ATP produced a transient facilitation of GDPs followed by a depressant effect. A similar biphasic effect was produced by blockade of the ectoATPase activity with 6‐N,N‐diethyl‐d‐β,γ‐dibromomethylene ATP (ARL‐67156). The effects of exogenous and endogenous ATP on GDPs were prevented by the P2X receptor antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS). On pyramidal cells, ATP upregulated spontaneous action‐potential‐dependent GABAA‐mediated synaptic events (GABA‐SPSPs), suggesting a network‐driven effect. Recordings from interneurones allowed comparison of ATP effects on GABAergic and glutamatergic synaptic activity. While ATP depressed GABA‐SPSPs via metabotropic P2Y1 receptors, it up‐ and downregulated glutamatergic SPSPs via PPADS‐sensitive receptors. Thus, ATP exerts an excitatory action on CA3 pyramidal cells via facilitation of GDPs and SPSPs. This excitatory drive is propagated to pyramidal cells by interneurons that represent the ‘common pathway’ for generation of GDPs and SPSPs. Our results show that ATP operating via distinct P2X and P2Y receptors directly contributes to modulate network activity at the early stages of postnatal development.
Role of extracellular Ca2+ in gating of CaV1.2 channels
Authors: Babich Olga , Isaev Dmytro , Shirokov Roman
Published: April 2005

We examined changes in ionic and gating currents in CaV1.2 channels when extracellular Ca2+ was reduced from 10 mm to 0.1 μm. Saturating gating currents decreased by two‐thirds (KD≈ 40 μm) and ionic currents increased 5‐fold (KD≈ 0.5 μm) due to increasing Na+ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca2+], which appeared to reflect the rapid activation of channels that were not blocked by Ca2+ and a slower reversal of Ca2+ blockade of the remaining channels. Removal of Ca2+ following inactivation of Ca2+ currents showed that Na+ currents were not affected by Ca2+‐dependent inactivation. Ca2+‐dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca2+ conductance and Ca2+‐dependent inactivation depend on extracellular Ca2+ and are linked to changes in selectivity.
Different kinetic properties of two T‐type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane
Authors: Joksovic Pavle M. , Bayliss Douglas A. , Todorovic Slobodan M.
Published: April 2005

Currents arising from T‐type Ca2+ channels in nucleus reticularis thalami (nRT) play a critical role in generation of low‐amplitude oscillatory bursting involving mutually interconnected cortical and thalamic neurones, and are implicated in the state of arousal and sleep, as well as seizures. Here we show in brain slices from young rats that two kinetically different T‐type Ca2+ currents exist in nRT neurones, with a slowly inactivating current expressed only on proximal dendrites, and fast inactivating current predominantly expressed on soma. Nickel was about twofold more potent in blocking fast (IC50 64 μm) than slow current (IC50 107 μm). The halogenated volatile anaesthetic enflurane blocked both currents, but only the slowly inactivating current was affected in voltage‐dependent fashion. Slow dendritic current was essential for generation of low‐threshold Ca2+ spikes (LTS), and both enflurane and nickel also suppressed LTS and neuronal burst firing at concentrations that blocked isolated T currents. Differential kinetic properties of T currents expressed in cell soma and proximal dendrites of nRT neurones indicate that various subcellular compartments may exhibit different membrane properties in response to small membrane depolarizations. Furthermore, since blockade of two different T currents in nRT neurones by enflurane and other volatile anaesthetics occurs within concentrations that are relevant during clinical anaesthesia, our findings suggest that these actions could contribute to some important clinical effects of anaesthetics.
Bottom-Up Ecosystem Trophic Dynamics Determine Fish Production in the Northeast Pacific
Authors: Ware Daniel M. , Thomson Richard E.
Published: April 2005

We addressed the question of bottom-up versus top-down control of marine ecosystem trophic interactions by using annual fish catch data and satellite-derived (SeaWiFS) chlorophyll a measurements for the continental margin of western North America. Findings reveal a marked alongshore variation in retained primary production that is highly correlated with the alongshore variation in resident fish yield. The highest productivity occurs off the coasts of Washington and southern British Columbia. Zooplankton data for coastal British Columbia confirm strong bottom-up trophic linkages between phytoplankton, zooplankton, and resident fish, extending to regional areas as small as 10,000 square kilometers.
Solar Wind Origin in Coronal Funnels
Authors: Tu Chuan-Yi , Zhou Cheng , Marsch Eckart , Xia Li-Dong , Zhao Liang , Wang Jing-Xiu , Wilhelm Klaus
Published: April 2005

The origin of the solar wind in solar coronal holes has long been unclear. We establish that the solar wind starts flowing out of the corona at heights above the photosphere between 5 megameters and 20 megameters in magnetic funnels. This result is obtained by a correlation of the Doppler-velocity and radiance maps of spectral lines emitted by various ions with the force-free magnetic field as extrapolated from photospheric magnetograms to different altitudes. Specifically, we find that Ne
7+
ions mostly radiate around 20 megameters, where they have outflow speeds of about 10 kilometers per second, whereas C
3+
ions with no average flow speed mainly radiate around 5 megameters. Based on these results, a model for understanding the solar wind origin is suggested.
Cladosporium Avr2 Inhibits Tomato Rcr3 Protease Required for
Cf-2
-Dependent Disease Resistance
Authors: Rooney Henrietta C. E. , van't Klooster John W. , van der Hoorn Renier A. L. , Joosten Matthieu H. A. J. , Jones Jonathan D. G. , de Wit Pierre J. G. M.
Published: April 2005

How plants recognize pathogens and activate defense is still mysterious. Direct interaction between pathogen avirulence (Avr) proteins and plant disease resistance proteins is the exception rather than the rule. During infection,
Cladosporium fulvum
secretes Avr2 protein into the apoplast of tomato leaves and, in the presence of the extracellular leucine-rich repeat receptor-like Cf-2 protein, triggers a hypersensitive response (HR) that also requires the extracellular tomato cysteine protease Rcr3. We show here that Avr2 binds and inhibits Rcr3 and propose that the Rcr3-Avr2 complex enables the Cf-2 protein to activate an HR.
Molecular Mechanism for Connective Tissue Destruction by Dipeptidyl Aminopeptidase IV Produced by the Periodontal PathogenPorphyromonas gingivalis
Authors: Kumagai Yumi , Yagishita Hisao , Yajima Ayako , Okamoto Tatsuya , Konishi Kiyoshi
Published: April 2005

Porphyromonas gingivalisis a pathogen associated with adult periodontitis. It produces dipeptidyl aminopeptidase IV (DPPIV), which may act as a virulence factor by contributing to the degradation of connective tissue. We investigated the molecular mechanism by which DPPIV contributes to the destruction of connective tissue. DPPIV itself did not show gelatinase or collagenase activity toward human type I collagen, but it promoted the activity of the host-derived matrix metalloproteinase 2 (MMP-2) (gelatinase) and MMP-1 (collagenase). DPPIV bound to fibronectin and mediated the adhesion ofP. gingivalisto fibronectin. Mutant DPPIV with catalytic Ser mutagenized to Ala (DPPSA) did not accelerate the degradation of collagen and gelatin by MMPs but retained fibronectin-binding activity. The adhesion of human gingival fibroblasts and NIH 3T3 cells to fibronectin was inhibited by DPPIV. Strain 4351ADPPSA exhibited an intermediate level of virulence in mice, between that of the strain expressing wild-type DPPIV (4351ADPP) and that of the strain harboring only the plasmid vector (4351AVEC). It is suggested that both activity promoting the degradation of collagen and gelatin and binding to fibronectin are required for full virulence. These results reveal novel biological functions of DPPIV and suggest a pathological role in the progression of periodontitis.
Physiological profile in relation to playing position of elite college Gaelic footballers
Authors: McIntyre M C , Hall M
Published: April 2005

Objective:To examine the physiological profile, and its relation to playing position, of elite college Gaelic footballers.

Method:The subjects were 28 elite Gaelic footballers (12 backs, 12 forwards, and four midfielders; mean (SD) age 21 (1.67) years), who won a major intervarsity tournament (Sigerson Cup) three times in succession.

Results:There was general similarity among the members of the team, probably the result of a typical, common training programme. The team means for stature (1.81 (0.05) m), body mass index (81.6 (6.5)) and percentage body fat (14.5 (3.1)%), power output by Wingate test (absolute power 912 (152) W or 10.72 (1.6) W/kg) and sit and reach test (22.3 (5.5) cm) displayed no significant differences when analysed according to playing position. However, midfielders did have significantly larger body mass than backs (p<0.05) and greater maximal oxygen consumption (p<0.01) and greater vertical jumping ability than backs and forwards (vertical jump power output, p<0.01; vertical jump, p<0.01). Midfielders also had greater absolute handgrip strength (p<0.01).

Conclusion:The differences exhibited by midfielders despite identical training suggests that they stem from physiological adaptation to competition rather than training.
Cadence selection affects metabolic responses during cycling and subsequent running time to fatigue
Authors: Vercruyssen F , Suriano R , Bishop D , Hausswirth C , Brisswalter J
Published: April 2005

Objectives:To investigate the effect of cadence selection during the final minutes of cycling on metabolic responses, stride pattern, and subsequent running time to fatigue.

Methods:Eight triathletes performed, in a laboratory setting, two incremental tests (running and cycling) to determine peak oxygen uptake (Vo2peak) and the lactate threshold (LT), and three cycle-run combinations. During the cycle-run sessions, subjects completed a 30 minute cycling bout (90% of LT) at (a) the freely chosen cadence (FCC, 94 (5) rpm), (b) the FCC during the first 20 minutes and FCC−20% during the last 10 minutes (FCC−20%, 74 (3) rpm), or (c) the FCC during the first 20 minutes and FCC+20% during the last 10 minutes (FCC+20%, 109 (5) rpm). After each cycling bout, running time to fatigue (Tmax) was determined at 85% of maximal velocity.

Results:A significant increase in Tmaxwas found after FCC−20% (894 (199) seconds) compared with FCC and FCC+20% (651 (212) and 624 (214) seconds respectively). Vo2, ventilation, heart rate, and blood lactate concentrations were significantly reduced after 30 minutes of cycling at FCC−20% compared with FCC+20%. A significant increase in Vo2was reported between the 3rd and 10th minute of all Tmaxsessions, without any significant differences between sessions. Stride pattern and metabolic variables were not significantly different between Tmaxsessions.

Conclusions:The increase in Tmaxafter FCC−20% may be associated with the lower metabolic load during the final minutes of cycling compared with the other sessions. However, the lack of significant differences in metabolic responses and stride pattern between the run sessions suggests that other mechanisms, such as changes in muscular activity, probably contribute to the effects of cadence variation on Tmax.
Physiological adaptations to soccer specific endurance training in professional youth soccer players
Authors: McMillan K , Helgerud J , Macdonald R , Hoff J
Published: April 2005

Background:Improved oxygen uptake improves soccer performance as regards distance covered, involvements with the ball, and number of sprints. Large improvements in oxygen uptake have been shown using interval running. A similar physiological load arising from interval running could be obtained using the soccer ball in training.

Objectives:The main aim was to study physiological adaptations to a 10 week high intensity aerobic interval training program performed by professional youth soccer players, using a soccer specific ball dribbling track.

Methods:Eleven youth soccer players with a mean (SD) age of 16.9 (0.4) years performed high intensity aerobic interval training sessions twice per week for 10 weeks in addition to normal soccer training. The specific aerobic training consisted of four sets of 4 min work periods dribbling a soccer ball around a specially designed track at 90–95% of maximal heart frequency, with a 3 min recovery jog at 70% of maximal heart frequency between intervals.

Results:Mean Vo2maximproved significantly from 63.4 (5.6) to 69.8 (6.6) ml kg−1min−1, or 183.3 (13.2) to 201.5 (16.2) ml kg−0.75min−1(p<0.001). Squat jump and counter movement jump height increased significantly from 37.7 (6.2) to 40.3 (6.1) cm and 52.0 (4.0) to 53.4 (4.2) cm, respectively (p<0.05). No significant changes in body mass, running economy, rate of force development, or 10 m sprint times occurred.

Conclusion:Performing high intensity 4 min intervals dribbling a soccer ball around a specially designed track together with regular soccer training is effective for improving the Vo2maxof soccer players, with no negative interference effects on strength, jumping ability, and sprinting performance.
Hepatocellular adenomas associated with anabolic androgenic steroid abuse in bodybuilders: a report of two cases and a review of the literature
Authors: Socas L , Zumbado M , Pérez-Luzardo O , Ramos A , Pérez C , Hernández J R , Boada L D
Published: April 2005

Anabolic androgenic steroids (AAS) are used illicitly at high doses by bodybuilders. The misuse of these drugs is associated with serious adverse effects to the liver, including cellular adenomas and adenocarcinomas. We report two very different cases of adult male bodybuilders who developed hepatocellular adenomas following AAS abuse. The first patient was asymptomatic but had two large liver lesions which were detected by ultrasound studies after routine medical examination. The second patient was admitted to our hospital with acute renal failure and ultrasound (US) studies showed mild hepatomegaly with several very close hyperecogenic nodules in liver, concordant with adenomas at first diagnosis. In both cases the patients have evolved favourably and the tumours have shown a tendency to regress after the withdrawal of AAS. The cases presented here are rare but may well be suggestive of the natural course of AAS induced hepatocellular adenomas. In conclusion, sportsmen taking AAS should be considered as a group at risk of developing hepatic sex hormone related tumours. Consequently, they should be carefully and periodically monitored with US studies. In any case, despite the size of the tumours detected in these two cases, the possibility of spontaneous tumour regression must also be taken in account.
Reliability and validity of a brief physical activity assessment for use by family doctors
Authors: Marshall A L , Smith B J , Bauman A E , Kaur S
Published: April 2005

Objective:To evaluate the reliability and validity of a brief physical activity assessment tool suitable for doctors to use to identify inactive patients in the primary care setting.

Methods:Volunteer family doctors (n  =  8) screened consenting patients (n  =  75) for physical activity participation using a brief physical activity assessment tool. Inter-rater reliability was assessed within one week (n  =  71). Validity was assessed against an objective physical activity monitor (computer science and applications accelerometer; n  =  42).

Results:The brief physical activity assessment tool produced repeatable estimates of “sufficient total physical activity”, correctly classifying over 76% of cases (κ 0.53, 95% confidence interval (CI) 0.33 to 0.72). The validity coefficient was reasonable (κ 0.40, 95% CI 0.12 to 0.69), with good percentage agreement (71%).

Conclusions:The brief physical activity assessment tool is a reliable instrument, with validity similar to that of more detailed self report measures of physical activity. It is a tool that can be used efficiently in routine primary healthcare services to identify insufficiently active patients who may need physical activity advice.
High strain mechanical loading rapidly induces tendon apoptosis: an ex vivo rat tibialis anterior model
Authors: Scott A , Khan K M , Heer J , Cook J L , Lian O , Duronio V
Published: April 2005

Background:The role of apoptosis, or programmed cell death, has only recently been explored in tendon.

Objective:To investigate the development of apoptosis after high strain loading of rat tendon.

Methods:The right tibialis anterior tendons of three rats were prepared for mechanical loading, and left tendons were prepared identically as non-loaded controls. Tendon was loaded with 20% strain for six hours using a 1 Hz longitudinal sine wave signal. The following were used to assess apoptosis: (a) a monoclonal mouse antibody (F7-26) to label single stranded DNA breaks; (b) a rabbit polyclonal antibody that specifically recognises the cleaved form of caspase-3.

Results:Light microscopy confirmed that the high strain protocol induced a stretch overload injury. Control tendons showed little or no staining with the F7-26 antibody, but the loaded tendons displayed numerous apoptotic cells. The percentage of apoptotic cells (20%) in the loaded tendon was significantly greater than in the control tendon (1%) (p  =  0.000). The labelled cells colocalised with abnormal nuclear morphology, including nuclear fragmentation. The staining against cleaved caspase-3 was positive in loaded tendons only, and localised both to nucleus and cytoplasm.

Conclusion:This experiment extends knowledge of human tendon apoptosis by showing that apoptosis can occur in response to short term, high strain mechanical loading. This is the first report of mechanical loading of intact tendon causing excessive apoptosis.
THE ISING MODEL STUDIED USING EVOLUTIONARY APPROACH
Authors: GWIZDAŁŁA TOMASZ M.
Published: April 2005

Evolutionary algorithms are very powerful techniques for the search of global minima. In this work we want to present the evolutionary approach to the one of the most fundamental problems of solid state magnetism: the Ising model. For the samples built in the most simple way, i.e. only from the ±1 spins, various temperature characteristics coming from the minimization of Gibbs free energy with entropy calculated from the pair approximation are shown. The calculations have been performed for samples of different magnitude which allowed the consideration of finite size effects.
POLYPYRROLE NANOWIRES GENERATION USING CHEMICAL SYNTHESIS THROUGH ION TRACK MEMBRANES
Authors: KUMAR SANJEEV , CHAKARVARTI S. K.
Published: April 2005

Polypyrrole nanocylinders were produced by non-galvanically (chemically) synthesizing polypyrrole within the pores of nanoporous polycarbonate ion track-etched membranes. The morphology of the nanowires was characterized by scanning electron microscopy. The electrical conductivity of the nanocylinders was calculated by leaving the nanocylinders fixed in the insulating template membrane and evaluating the transmembrane resistance.
Lipopolysaccharide-induced liver injury in rats treated with the CYP2E1 inducer pyrazole
Authors: Lu Yongke , Wang Xiaodong , Cederbaum Arthur I.
Published: April 2005

Elevated LPS and elevated cytochrome P-450 2E1 (CYP2E1) in liver are two major independent risk factors in alcoholic liver disease. We investigated possible synergistic effects of the two risk factors in causing oxidative stress and liver injury. Sprague-Dawley rats were injected intraperitoneally with pyrazole (inducer of CYP2E1) for 2 days, and then LPS was injected via tail vein. Other rats were treated with pyrazole alone or LPS alone or saline. Eight hours later, blood was collected and livers were excised. Pathological evaluation showed severe inflammatory responses and necroses only in liver sections from rats in the pyrazole plus LPS group; blood transaminase levels were significantly elevated only in the combination group. Activities of caspase-3 and -9 and positive terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were highest in the LPS alone and the LPS plus pyrazole group, with no significant difference between the two groups. Lipid peroxidation and protein carbonyls in liver homogenate as well as in situ superoxide production were maximally elevated in the LPS plus pyrazole group. Levels of nitrite plus nitrate and inducible nitric oxide (NO) synthase (iNOS) content were comparably elevated in LPS alone and the LPS plus pyrazole group; however, 3-nitrotyrosine adducts were elevated in the combined group but not the LPS group. It is likely that LPS induction of iNOS, which produces NO, coupled to pyrazole induction of CYP2E1 which produces superoxide, sets up conditions for maximal peroxynitrite formation and production of 3-nitrotyrosine adducts. CYP2E1 activity and content were elevated in the pyrazole and the LPS plus pyrazole groups. Immunohistochemical staining indicated that distribution of CYP2E1 was in agreement with that of necrosis and production of superoxide. These results show that pyrazole treatment enhanced LPS-induced necrosis, not apoptosis. The enhanced liver necrosis appears to involve an increase in oxidative and nitrosative stress generated by the combination of LPS plus elevated CYP2E1 levels.
Grp78, Grp94, and Grp170 interact with α1-antitrypsin mutants that are retained in the endoplasmic reticulum
Authors: Schmidt Bela Z. , Perlmutter David H.
Published: April 2005

In α1-antitrypsin (α1-AT) deficiency, a mutant form of α1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant α1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The α1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which α1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant α1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of ∼110–130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic α1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the α1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that ∼85% of α1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and ∼15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of α1-AT Z as well as on its residual secretion.
Transport of extracellular l-arginine via cationic amino acid transporter is required during in vivo endothelial nitric oxide production
Authors: Zani Brett G. , Bohlen H. Glenn
Published: April 2005

In cultured endothelial cells, 70–95% of extracellular l-arginine uptake has been attributed to the cationic amino acid transporter-1 protein (CAT-1). We tested the hypothesis that extracellular l-arginine entry into endothelial cells via CAT-1 plays a crucial role in endothelial nitric oxide (NO) production during in vivo conditions. Using l-lysine, the preferred amino acid transported by CAT-1, we competitively inhibited extracellular l-arginine transport into endothelial cells during conditions of NaCl hyperosmolarity, low oxygen, and flow increase. Our prior studies indicate that each of these perturbations causes NO-dependent vasodilation. The perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature. Suppression of extracellular l-arginine transport significantly and strongly inhibited increases in vascular [NO] and intestinal blood flow during NaCl hyperosmolarity, lowered oxygen tension, and increased flow. These results suggest that l-arginine from the extracellular space is accumulated by CAT-1. When CAT-1-mediated transport of extracellular l-arginine into endothelial cells was suppressed, the endothelial cell NO response to a wide range of physiological stimuli was strongly depressed.
Sympathetic α-adrenergic regulation of blood flow and volume in hamsters arousing from hibernation
Authors: Osborne P. G. , Sato J. , Shuke N. , Hashimoto M.
Published: April 2005

Mammals arousing from hibernation display pronounced regional heterothermy, where the thoracic and head regions warm faster than the abdominal and hindlimb regions. We used laser-Doppler flowmetry to measure peripheral hind foot blood flow during hibernation and arousal and gamma imaging of technetium-labeled albumin to measure whole blood volume distribution in hamsters arousing from hibernation. It was discovered that the hibernating hamster responds to physical but not to sound or hypercapnic stimulation with rapid, 73% reduction of hind foot blood flow. Hind foot blood flow vasoconstriction was maintained from the onset of arousal until late in arousal when rectal temperature was rapidly increased. α-Adrenergic blockade early in arousal increased hind foot blood flow by 700%, suggesting that vasoconstriction was mediated by activation of sympathetic tone. Gamma imaging revealed that, by the early phase of arousal from hibernation, the blood volume of the body below the liver is greatly reduced, whereas blood volumes of the thorax and head are much greater than corresponding volumes in anesthetized hamsters. As arousal progresses and cardiac activity increases and regional heterothermy develops, this regional blood volume distribution is largely maintained; however, blood volume slowly decreases in the thoracic region and slowly increases in the shoulder and head regions. The rapid increase in rectal temperature, characteristic of mid- to late- arousal phases, is probably mediated, in part, by reduction of adrenergic tone on abdominal and hindlimb vasculature. Warm blood then moves into the hind body, produces an increase in temperature, blood flow, and blood volume in the hind body and compensatory reductions of blood volume in the neck, head, and thoracic regions.