bluuebunny/crossref_metadata_2025 huggingface dataset:
Stabilities and activities of the N‐ and C‐domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli
Authors: Suzuki Yutaka , Takano Kazufumi , Kanaya Shigenori
Published: January 2005

FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1, is a dimeric protein with peptidyl prolyl cis‐trans isomerase (PPIase) activity. According to homology modeling, it consists of an N‐terminal domain, which is involved in dimerization of the protein, and a C‐terminal catalytic domain. A long α3 helix spans these domains. An N‐domain with the entire α3 helix (N‐domain+) and a C‐domain with the entire α3 helix (C‐domain+) were overproduced in Escherichia coli in a His‐tagged form, purified, and their biochemical properties were compared with those of the intact protein. C‐domain+ was shown to be a monomer and enzymatically active. Its optimum temperature for activity (10 °C) was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N‐domain is involved in substrate‐binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5 °C and 46.6 °C for the intact protein, and single heat absorption peaks at 44.7 °C for N‐domain+ and 35.6 °C for C‐domain+. These results indicate that the thermal unfolding transitions of the intact protein at lower and higher temperatures represent those of C‐ and N‐domains, respectively. Because the unfolding temperature of C‐domain+ is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibility around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22.
Mammalian transglutaminases
Authors: Esposito Carla , Caputo Ivana
Published: January 2005

Transglutaminases form a large family of intracellular and extracellular enzymes that catalyse the Ca2+‐dependent post‐translational modification of proteins. Despite significant advances in our understanding of the biological role of most mammalian transglutaminase isoforms, recent findings suggest new scenarios, most notably for the ubiquitous tissue transglutaminase. It is becoming apparent that some transglutaminases, normally expressed at low levels in many tissue types, are activated and/or overexpressed in a variety of diseases, thereby resulting in enhanced concentrations of cross‐linked proteins. As applies to all enzymes that exert their metabolic function by modifying the properties of target proteins, the identification and characterization of the modified proteins will cast light on the functions of transglutaminases and their involvement in human diseases. In this paper we review data on the properties of mammalian transglutaminases, particularly as regards their protein substrates and the relevance of transglutaminase‐catalysed reactions in physiological and disease conditions.
Stimulation of p‐nitrophenylphosphatase activity of Na+/K+‐ATPase by NaCl with oligomycin or ATP
Authors: Homareda Haruo , Ushimaru Makoto
Published: January 2005

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain‐sensitive and K+‐dependent p‐nitrophenylphosphatase (pNPPase) activity of Na+/K+‐ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+‐binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+‐ATPase by p‐nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+‐occluded E1 state of Na+/K+‐ATPase, it seemed that the Na+‐occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high‐energy phosphoenzyme formed from ATP with Na+, is also the Na+‐occluded E1 state, it is suggested that the Na+‐occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between α subunits. Therefore, membrane‐bound Na+/K+‐ATPase would function as at least an (αβ)2‐diprotomer with interacting α subunits at the phosphorylation step.
Characterization of the tRNA and ribosome‐dependent pppGpp‐synthesis by recombinant stringent factor from Escherichia coli
Authors: Knutsson Jenvert Rose‐Marie , Holmberg Schiavone Lovisa
Published: January 2005

Stringent factor is a ribosome‐dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino‐acyl site (A‐site) but it is unclear how activation occurs. A His‐tagged stringent factor was isolated by affinity‐chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome‐dependent activity that was inhibited by the A‐site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA‐ and ribosome‐dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A‐site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A‐site. However, tRNA‐saturation curves, performed at two different ribosome/stringent factor ratios and filter‐binding assays, did not support this hypothesis.
Hidden stereospecificity in the biosynthesis of divinyl ether fatty acids
Authors: Hamberg Mats
Published: January 2005

Incubations of [8(R)‐2H]9(S)‐hydroperoxy‐10(E),12(Z)‐octadecadienoic acid, [14(R)‐2H]13(S)‐hydroperoxy‐9(Z),11(E)‐octadecadienoic acid and [14(S)‐2H]13(S)‐hydroperoxy‐9(Z),11(E)‐octadecadienoic acid were performed with preparations of plant tissues containing divinyl ether synthases. In agreement with previous studies, generation of colneleic acid from the 8(R)‐deuterated 9(S)‐hydroperoxide was accompanied by loss of most of the deuterium label (retention, 8%), however, the opposite result (98% retention) was observed in the generation of 8(Z)‐colneleic acid from the same hydroperoxide. Formation of etheroleic acid and 11(Z)‐etheroleic acid from the 14(R)‐deuterated 13(S)‐hydroperoxide was accompanied by loss of most of the deuterium (retention, 7–8%), and, as expected, biosynthesis of these divinyl ethers from the corresponding 14(S)‐deuterated hydroperoxide was accompanied by retention of deuterium (retention, 94–98%). Biosynthesis of ω5(Z)‐etheroleic acid from the 14(R)‐ and 14(S)‐deuterated 13(S)‐hydroperoxides showed the opposite results, i.e. 98% retention and 4% retention, respectively. The experiments demonstrated that biosynthesis of divinyl ether fatty acids from linoleic acid 9‐ and 13‐hydroperoxides takes place by a mechanism that involves stereospecific abstraction of one of the two hydrogen atoms α to the hydroperoxide carbon. Furthermore, a consistent relationship between the absolute configuration of the hydrogen atom eliminated (R or S) and the configuration of the introduced vinyl ether double bond (E or Z) emerged from these results. Thus, irrespective of which hydroperoxide regioisomer served as the substrate, divinyl ether synthases abstracting the pro‐R hydrogen generated divinyl ethers having an E vinyl ether double bond, whereas enzymes abstracting the pro‐S hydrogen produced divinyl ethers having a Z vinyl ether double bond.
Binding of non‐natural 3′‐nucleotides to ribonuclease A
Authors: Jenkins Cara L. , Thiyagarajan Nethaji , Sweeney Rozamond Y. , Guy Michael P. , Kelemen Bradley R. , Acharya K. Ravi , Raines Ronald T.
Published: January 2005

2′‐Fluoro‐2′‐deoxyuridine 3′‐phosphate (dUFMP) and arabinouridine 3′‐phosphate (araUMP) have non‐natural furanose rings. dUFMP and araUMP were prepared by chemical synthesis and found to have three‐ to sevenfold higher affinity than uridine 3′‐phosphate (3′‐UMP) or 2′‐deoxyuridine 3′‐phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3′‐UMP, dUFMP, and araUMP (pKa = 5.9) being more anionic than that of dUMP (pKa = 6.3). The three‐dimensional structures of the crystalline complexes of RNase A with dUMP, dUFMP and araUMP were determined at < 1.7 Å resolution by X‐ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3′‐UMP and dUFMP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A·araUMP complex, the 2′‐hydroxyl group is exposed to the solvent. All four 3′‐nucleotides bind more tightly to wild‐type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3′‐nucleotides, and indicate that non‐natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.
Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues
Authors: Liang Yanbin , Li Chen , Guzman Victor M. , Chang William W. , Evinger Albert J. , Sao Dyna , Woodward David F.
Published: January 2005

Regulator of G protein signaling (RGS) proteins act as GTPase‐activating proteins (GAPs) for Gα subunits and negatively regulate G protein‐coupled receptor signaling. Using RGS5 gene‐specific RT‐PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N‐terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N‐terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Gαi coupled cannibinoid receptor‐1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Gαq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Gαq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.
A novel cholesterol‐based detergent
Authors: Gehrig‐Burger Katja , Kohout Ladislav , Gimpl Gerald
Published: January 2005

Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol‐based detergent developed for functional solubilization of cholesterol‐dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein‐coupled receptor requiring cholesterol for its high‐affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6‐azocholestan‐3β‐ol[3αH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol‐binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol‐binding proteins. Similar to Triton X‐100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e. detergent‐insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol‐dependent membrane proteins and isolation of rafts.
Two beta‐alanyl‐CoA:ammonia lyases in Clostridium propionicum
Authors: Herrmann Gloria , Selmer Thorsten , Jessen Holly J. , Gokarn Ravi R. , Selifonova Olga , Gort Steve J. , Buckel Wolfgang
Published: January 2005

The fermentation of β‐alanine by Clostridium propionicum proceeds via activation to the CoA‐thiol ester, followed by deamination to acryloyl‐CoA, which is also an intermediate in the fermentation of l‐alanine. By shifting the organism from the carbon and energy source α‐alanine to β‐alanine, the enzyme β‐alanyl‐CoA:ammonia lyase is induced 300‐fold (≈ 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on β‐alanine. The deduced β‐alanyl‐CoA:ammonia lyase proteins are related to putative β‐aminobutyryl‐CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. β‐Alanyl‐CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent Km value for acryloyl‐CoA was measured as 23 ± 4 µm, independent of the concentration of the second substrate ammonia; kcat/Km was calculated as 107 m−1·s−1. The apparent Km for ammonia was much higher, 70 ± 5 mm at 150 µm acryloyl‐CoA with a much lower kcat/Km of 4 × 103 m−1·s−1. In the reverse reaction, a Km of 210 ± 30 µM was obtained for β‐alanyl‐CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of β‐alanyl‐CoA:ammonia lyase in β‐alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl‐CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between l‐alanine/β‐alanine or d‐lactate/3‐hydroxypropionate.
Identification of a domain in the α‐subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the γ‐subunit
Authors: Dahinden Pius , Pos Klaas M. , Dimroth Peter
Published: January 2005

The oxaloacetate decarboxylase Na+ pumps OAD‐1 and OAD‐2 of Vibrio cholerae are composed of a peripheral α‐subunit associated with two integral membrane‐bound subunits (β and γ). The α‐subunit contains the carboxyltransferase domain in its N‐terminal portion and the biotin‐binding domain in its C‐terminal portion. The γ‐subunit plays a profound role in the assembly of the complex. It interacts with the β‐subunit through its N‐terminal membrane‐spanning region and with the α‐subunit through its hydrophilic C‐terminal domain. The biochemical and structural requirements for the latter interaction were analysed with OAD‐2 expression clones for subunit α‐2 and the C‐terminal domain of γ‐2, termed γ′‐2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of α‐2 was identified by deletion mutagenesis to be essential for the binding with γ′‐2. This portion of the α‐subunit is connected via flexible linker peptides to the carboxyltransferase domain at its N terminus and to the biotin‐binding domain at its C terminus. Results of site‐directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the stability of the complex with γ′‐2. This peptide therefore represents a newly identified, separate domain of the α‐subunit and has been called the ‘association domain’.
Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity
Authors: Willemoës Martin , Mølgaard Anne , Johansson Eva , Martinussen Jan
Published: January 2005

GTP is an allosteric activator of CTP synthase and acts to increase the kcat for the glutamine‐dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy‐anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy‐anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy‐anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild‐type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10‐ to 50‐fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four‐ to 10‐fold increases in KA for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild‐type enzyme. The elevated KA for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy‐anion hole of the GATase domain.
An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal‐binding domains of the Menkes protein
Authors: Banci Lucia , Bertini Ivano , Ciofi‐Baffoni Simone , Chasapis Christos T , Hadjiliadis Nick , Rosato Antonio
Published: January 2005

The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper‐transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)–MNK2/MNK5. The solution structures of both apo‐ and copper(I)‐MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.
New insights into Fragile X syndrome
Authors: Pozdnyakova Irina , Regan Lynne
Published: January 2005

Lack of functional Fragile X mental retardation protein (FMRP) is the primary cause of the Fragile‐mental retardation syndrome in humans. In most cases, the disease results from transcriptional silencing of fragile mental retardation gene 1, fmr1, which encodes FMRP. However, a single missense mutation (I304N) in the second KH domain of FMRP gives rise to a particularly severe case of Fragile X syndrome. A Drosophila homolog of FMRP has been identified, Drosophila Fragile X related protein (dFXRP). The corresponding missense mutation in dFXRP, the I307N, has pronounced effects on the in vivo activity of the protein. The effect of the point mutation on the structure and function of FMRP is unclear, and published data are contradictory. No in vitro structural or stability studies have been performed on dFXRP. Here we show that a construct that contains only the tandem KH1‐KH2 domains is a stable, well‐folded unit suitable for detailed structural and functional characterization. Using this KH1‐KH2 construct we explicitly test a hypothesis that has been proposed to explain the effect of the Ile→Asn mutation: that it causes complete unfolding of the protein. Here we show that the I307N point mutation does not completely unfold the KH domain. The KH1‐KH2 construct bearing I307N substitution is stable in isolation and adopts a native‐like fold. Thus our data favor alternative explanations for the in vivo observed loss of dFXRP activity associated with I307N mutation: (a) the point mutation might affect intra and/or inter‐molecular interactions of dFXRP; or (b) it might impair dFXRP's interactions with its RNA target(s).
Active, non‐spring‐like muscle movements in human postural sway: how might paradoxical changes in muscle length be produced?
Authors: Loram Ian D. , Maganaris Constantinos N. , Lakie Martin
Published: January 2005

In humans, during standing the calf muscles soleus and gastrocnemius actively prevent forward toppling about the ankles. It has been generally assumed that these postural muscles behave like springs with dynamic stiffness reflecting their mechanical properties, reflex gain including higher derivatives, and central control. Here, for the first time, we have used an ultrasound scanner and automated image analysis to record the tiny muscular movements occurring in normal standing. This new, non‐invasive technique resolves changes in muscle length as small as 10 μm without disturbing the standing process. This technical achievement has allowed us to test the long‐established mechano‐reflex, muscle spring hypothesis that muscle length changes in a spring‐like way during sway of the body. Our results contradict that hypothesis. Muscle length changes in a non‐spring‐like manner: on average, shortening during forward sway and lengthening during backwards sway (paradoxical movements). This counter‐intuitive result is a consequence of the fact that calf muscles generate tension through a series elastic component (SEC, Achilles tendon and foot) which limits maximal ankle stiffness to 92 ± 20% of that required to balance the body. Paradoxical movements cannot be generated by stretch reflexes with constant intrafusal drive but might be produced by reflex coupling of extrafusal (α) and intrafusal (β, γ) drive or by positive force feedback. Standing requires the predictive ability to produce the observed muscle movements preceded (110 ± 50 ms) by corresponding changes in integrated EMG signal. We suggest higher level anticipatory control is more plausible.
Human postural sway results from frequent, ballistic bias impulses by soleus and gastrocnemius
Authors: Loram Ian D. , Maganaris Constantinos N. , Lakie Martin
Published: January 2005

It has been widely assumed for nearly a century, that postural muscles operate in a spring‐like manner and that muscle length signals joint angle (the mechano‐reflex mechanism). Here we employ automated analysis of ultrasound images to resolve calf muscle (soleus and gastrocnemius) length changes as small as 10 μm in standing subjects. Previously, we have used balancing of a real inverted pendulum to make predictions about human standing. Here we test and confirm these predictions on 10 subjects standing quietly. We show that on average the calf muscles are actively adjusted 2.6 times per second and 2.8 times per unidirectional sway of the body centre of mass (CoM). These alternating, small (30–300 µm) movements provide impulsive, ballistic regulation of CoM movement. The timing and pattern of these adjustments are consistent with multisensory integration of all information regarding motion of the CoM, pattern recognition, prediction and planning using internal models and are not consistent with control solely by local reflexes. Because the system is unstable, errors in stabilization provide a perturbation which grows into a sway which has to be reacted to and corrected. Sagittal sway results from this impulsive control of calf muscle activity rather than internal sources (e.g. the heart, breathing). This process is quite unlike the mechano‐reflex paradigm. We suggest that standing is a skilled, trial and error activity that improves with experience and is automated (possibly by the cerebellum). These results complement and extend our recent demonstration that paradoxical muscle movements are the norm in human standing.
Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration
Authors: Liu Ruisheng , Persson A. Erik G.
Published: January 2005

Cell volume and cytosolic Ca2+ concentration ([Ca2+]i) were measured in rabbit macula densa (MD) cells loaded with calcein and Fura Red using confocal microscopy. [Ca2+]i was also analysed with Indo‐1 and fura‐2. We used isolated microperfused thick ascending limbs with attached glomerulus. The results showed that when the luminal NaCl concentration (‘NaCl’) was decreased from 35 to 10 mm, the cell volume decreased by 10.4%, and [Ca2+]i increased by 9.5%. This increase was inhibited in Ca2+‐free solution. When luminal [NaCl] was changed from 35 to 135 mm, the cell volume increased by 15.1%, and [Ca2+]i did not change. The cell volume alterations were not different in Ca2+‐free solutions. Using Indo‐1, basal [Ca2+]i in MD cells was 107.8 nm. When luminal [NaCl] was changed from 135 to 10 mm, [Ca2]i increased by 23.5 nm. Using fura‐2, the basal [Ca2+]i in MD cells was 115.3 nm, and when luminal [NaCl] was changed from 135 or 35 to 10 mm, [Ca2+]i change was 30.1 or 10.6 nm, respectively. An increase in [NaCl] caused no change in [Ca2+]i. In Ca2+‐free solution, no change in [Ca2+]i occurred. A stepwise decrease in luminal [NaCl] resulted in a sigmoid increase in [Ca2+]i in MD cells. The steepest part of the curve was between 70 and 10 mm. In conclusion, we found that MD cells have cell volume regulation, and that [Ca2+]i elevation caused by decreased luminal [NaCl] is independent of the cell volume.
Reduced ventricular flow propagation velocity in elite athletes is augmented with the resumption of exercise training
Authors: Naylor Louise H. , Arnolda Leonard F. , Deague Jenny A. , Playford David , Maurogiovanni Antonio , O'Driscoll Gerard , Green Daniel J.
Published: January 2005

Chronic exercise induces physiological enlargement of the left ventricle (‘athlete's heart’), but the effects of current and long‐term exercise training on diastolic function have not been investigated. Echocardiography and Doppler imaging were used to assess left ventricular (LV) dimensions and indices of diastolic filling in 22 elite athletes at the end of their ‘off‐season’ (baseline) and, subsequently, following 3 and 6 months of training. Twelve matched controls were also studied at baseline, 3 and 6 months. Compared to controls at baseline, athletes exhibited significantly higher LV mass (235.7 ± 7.1 g versus 178.1 ± 14.5 g, P < 0.01) and reduced flow propagation velocity (VP: 50.21 ± 1.7 versus 72.2 ± 3.6 cm s−1, P < 0.01), a measure of diastolic function. Three months of training further increased LV mass in athletes (253.2 ± 7.1 g; P < 0.01 versus baseline), and significantly increased their VP (66.7 ± 2.5 cm s−1; P < 0.05 versus baseline). These trends for increased mass and diastolic filling persisted following 6 months of training (LV mass 249.0 ± 8.7 g P < 0.05 versus baseline; VP 75.7 ± 3.0 cm s−1; P < 0.01 versus baseline, and P= 0.01 versus 3 months). This study suggests that following a period of relative inactivity the rate of ventricular relaxation during early diastole may be slowed in athletes who exhibit ventricular hypertrophy, whilst resumption of training increases the speed of ventricular relaxation in the presence of further hypertrophy of the left ventricle.
Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation
Authors: Mühlhäusler B. S. , Adam C. L. , Marrocco E. M. , Findlay P. A. , Roberts C. T. , McFarlane J. R. , Kauter K. G. , McMillen I. C.
Published: January 2005

In the present study, our aim was to determine whether intrafetal glucose infusion increases fetal adiposity, synthesis and secretion of leptin and regulates gene expression of the ‘appetite regulatory’ neuropeptides neuropepetide Y (NPY), agouti‐related peptide (AGRP), pro‐opiomelanocortin (POMC) and cocaine‐ and amphetamine‐regulated transcript (CART) and receptors (leptin receptor (OB‐Rb) and melancortin 3 receptor (MC3R)) within the fetal hypothalamus. Glucose (50% dextrose in saline) or saline was infused (7.5 ml h−1) into fetal sheep between 130 and 140 days gestation (term = 150 ± 3 days gestation). Glucose infusion increased circulating glucose and insulin concentrations, mean lipid locule size (532.8 ± 3.3 μm2versus 456.7 ± 14.8 μm2) and total unilocular fat mass (11.7 ± 0.6 g versus 8.9 ± 0.6 g) of the perirenal fat depot. The expression of OB‐Rb mRNA was higher in the ventromedial nucleus compared to the arcuate nucleus of the hypothalamus in both glucose and saline infused fetuses (F= 8.04; P < 0.01) and there was a positive correlation between expression of OB‐Rb and MC3R mRNA in the arcuate nucleus (r= 0.81; P < 0.005). Glucose infusion increased mRNA expression for POMC, but not for the anorectic neuropeptide CART, or the orexigenic neuropeptides NPY and AGRP, in the arcuate nucleus of the fetal hypothalamus. These findings demonstrate that increased circulating glucose and insulin regulate gene expression of the neuropeptides within the fetal hypothalamus that are part of the neural network regulating energy balance in adult life.
Induced sharp wave‐ripple complexes in the absence of synaptic inhibition in mouse hippocampal slices
Authors: Nimmrich Volker , Maier Nikolaus , Schmitz Dietmar , Draguhn Andreas
Published: January 2005

The characteristic, behaviour‐related network oscillations of the mammalian hippocampus (θ, γ and ripples) are accompanied by strongly phase‐coupled action potentials in specific subsets of GABAergic interneurones. It has been suggested that the resulting phasic, repetitive inhibition shapes rhythmic coherent activity of the neuronal network. Here, we examined whether synaptic inhibition entrains ∼200 Hz network ripples by applying the GABAA receptor antagonist gabazine to CA1 minislices of mouse hippocampus. Gabazine blocked spontaneously occurring sharp wave–ripple (SPW–R) activity. However, local application of KCl to the dendritic layer elicited excitatory sharp waves on which ∼200 Hz ripple oscillations were superimposed with equal temporal properties of native SPW–R. The activity also persisted in the additional presence of blockers of glutamatergic synaptic transmission. In contrast, synchrony was largely abolished after addition of gap junction blockers. Thus, GABAergic transmission appears to be involved in the generation of sharp waves but phasic inhibition is no prerequisite for the precise synchronization of hippocampal neurones during high‐frequency oscillations at ∼200 Hz. Gap junctions on the other hand seem to be necessary to orchestrate coordinated activity within the ripple frequency domain.
Carbon dioxide sensitivity during hypoglycaemia‐induced, elevated metabolism in the anaesthetized rat
Authors: Bin‐Jaliah I. , Maskell P. D. , Kumar P.
Published: January 2005

We have utilized an anaesthetized rat model of insulin‐induced hypoglycaemia to test the hypothesis that peripheral chemoreceptor gain is augmented during hypermetabolism. Insulin infusion at 0.4 U kg −1min−1 decreased blood glucose concentration significantly to 3.37 ± 0.12 mmol l−1. Whole‐body metabolism and basal ventilation were elevated without increase in P (altered non‐significantly from the control level, to 37.3 ± 2.6 mmHg). Chemoreceptor gain, measured either as spontaneous ventilatory airflow sensitivity to P during rebreathing, or by phrenic minute activity responses to altered P induced by varying the level of artificial ventilation, was doubled during the period of hypermetabolism. This stimulatory effect was primarily upon the mean inspiratory flow rate, or phrenic ramp component of breathing and was reduced by 75% following bilateral carotid sinus nerve section. In vitro recordings of single carotid body chemoafferents showed that reducing superfusate glucose concentration from 10 mm to 2 mm reduced CO2 chemosensitivity significantly from 0.007 ± 0.002 Hz mmHg−1 to 0.001 ± 0.002 Hz mmHg−1. Taken together, these data suggest that the hyperpnoea observed during hypermetabolism might be mediated by an increase in the CO2 sensitivity of the carotid body, and this effect is not due to the insulin‐induced fall in blood glucose concentration.
Reduced glycine transporter type 1 expression leads to major changes in glutamatergic neurotransmission of CA1 hippocampal neurones in mice
Authors: Martina Marzia , B.‐Turcotte Marie‐Eve , Halman Samantha , Tsai Guochuan , Tiberi Mario , Coyle Joseph T. , Bergeron Richard
Published: January 2005

To investigate the effects of persistent elevation of synaptic glycine at Schaffer collateral–CA1 synapses of the hippocampus, we studied the glutamatergic synaptic transmission in acute brain slices from mice with reduced expression of glycine transporter type 1 (GlyT1+/−) as compared to wild type (WT) littermates using whole‐cell patch‐clamp recordings of CA1 pyramidal cells. We observed faster decay kinetics, reduced ifenprodil sensitivity and increased zinc‐induced antagonism in N‐methyl‐d‐aspartate receptor (NMDAR) currents of GlyT1+/− mice. Moreover, the ratio α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor (AMPAR)/NMDAR was decreased in mutants compared to WT. Surprisingly, this change was associated with a reduction in the number of AMPARs expressed at the CA1 synapses in the mutants compared to WT. Overall, these findings highlight the importance of GlyT1 in regulating glutamatergic neurotransmission.
Mechanisms of acetylcholine‐mediated vasodilatation in young and aged human skin
Authors: Holowatz Lacy A. , Thompson Caitlin S. , Minson Christopher T. , Kenney W. Larry
Published: January 2005

Thermoregulatory cutaneous vasodilatation (VD) is attenuated in aged skin. While acetylcholine (ACh) plays a role in thermally mediated VD, the precise mechanisms through which ACh‐mediated VD acts and whether those downstream mechanisms change with ageing are unclear. We tested the hypotheses that both nitric oxide (NO)‐ and prostanoid‐mediated pathways contribute to exogenous ACh‐mediated VD, and that both are attenuated with advanced age. Twelve young (Y: 23 ± 1 years) and 10 older (O: 69 ± 1 years) subjects underwent infusions of 137.5 μmACh at four intradermal microdialysis sites: control (C, Ringer solution), NO synthase inhibited (NOS‐I, 10 mml‐NAME), cyclooxygenase inhibited (COX‐I, 10 mmketorolac) and NOS‐I + COX‐I. Red blood cell flux was monitored using laser‐Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated (laser‐Doppler flux/mean arterial pressure) and normalized to maximal CVC (%CVCmax) (28 mmsodium nitroprusside + local heating to 43°C). Baseline %CVCmaxwas increased in the O at COX‐I sites (COX‐I 16 ± 1, NOS‐I + COX‐I 16 ± 2versusC 10 ± 1%CVCmax;P< 0.001) but not in the young, suggesting an age‐related shift toward COX vasoconstrictors contributing to basal cutaneous vasomotor tone. There was no difference in peak %CVCmaxduring ACh infusion between age groups, and the response was unchanged by NOS‐I (O: NOS‐I 35 ± 5versusC 38 ± 5%CVCmax;P= 0.84) (Y: NOS‐I 41 ± 4versusC 39 ± 4%CVCmax;P= 0.67). COX‐I and NOS‐I + COX‐I attenuated the peak CVC response to ACh in both groups (COX‐I O: 29 ± 3, Y: 22 ± 2%CVCmaxversusC;P< 0.001 both groups; NOS‐I + COX‐I O: 32 ± 3versusY: 29 ± 2%CVCmax;versusC;P< 0.001 both groups). ACh mediates cutaneous VD through prostanoid and non‐NO‐, non‐prostanoid‐dependent pathways. Further, older subjects have a diminished prostanoid contribution to ACh‐mediated VD.
The physiological regulation of toll‐like receptor expression and function in humans
Authors: Lancaster Graeme I. , Khan Qamar , Drysdale Pam , Wallace Fiona , Jeukendrup Asker E. , Drayson Mark T. , Gleeson Michael
Published: January 2005

Eleven mammalian toll‐like receptors (TLRs 1–11) have been identified to date and are known to play a crucial role in the regulation of immune responses; however, the factors that regulate TLR expression and function in vivo are poorly understood. Therefore, in the present study, we investigated the physiological regulation of TLR expression and function in humans. To examine the influence of diurnal rhythmicity on TLR expression and function, peripheral venous blood samples were collected from healthy volunteers (n= 8) at time points coinciding with the peak and nadir in the endogenous circulating cortisol concentration. While no diurnal rhythmicity in the expression of TLRs 1, 2, 4 or 9 was observed, the upregulation of costimulatory (CD80 and CD86) and antigen‐presenting (MHC class II) molecules on CD14+ monocytes following activation with specific TLR ligands was greater (P < 0.05) in samples obtained in the evening compared with the morning. To examine the influence of physical stress on TLR expression and function, peripheral venous blood samples were collected from healthy volunteers (n= 11) at rest and following 1.5 h of strenuous exercise in the heat (34°C). Strenuous exercise resulted in a decrease (P < 0.005) in the expression of TLRs 1, 2 and 4 on CD14+ monocytes. Furthermore, the upregulation of CD80, CD86, MHC class II and interleukin‐6 by CD14+ monocytes following activation with specific TLR ligands was decreased (P < 0.05) in samples obtained following exercise compared with at rest. These results demonstrate that TLR function is subject to modulation under physiological conditions in vivo and provide evidence for the role of immunomodulatory hormones in the regulation of TLR function.
Chronic Lymphocytic Inflammation Specifies the Organ Tropism of Prions
Authors: Heikenwalder Mathias , Zeller Nicolas , Seeger Harald , Prinz Marco , Klöhn Peter-Christian , Schwarz Petra , Ruddle Nancy H. , Weissmann Charles , Aguzzi Adriano
Published: January 2005

Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1
+
cells expressing the normal cellular prion protein PrP
C
. By contrast, inflamed organs of mice lacking lymphotoxin-α or its receptor did not accumulate the abnormal isoform PrP
Sc
, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.
Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain Barrier
Authors: Grab Dennis J. , Perides George , Dumler J. Stephen , Kim Kee Jun , Park Jinho , Kim Yuri V. , Nikolskaia Olga , Choi Kyoung Seong , Stins Monique F. , Kim Kwang Sik
Published: January 2005

Neurological manifestations of Lyme disease in humans are attributed in part to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system (CNS) byBorrelia burgdorferi. However, how the spirochetes cross the BBB remains an unresolved issue. We examined the traversal ofB. burgdorferiacross the human BBB and systemic endothelial cell barriers using in vitro model systems constructed of human brain microvascular endothelial cells (BMEC) and EA.hy 926, a human umbilical vein endothelial cell (HUVEC) line grown on Costar Transwell inserts. These studies showed thatB. burgdorferidifferentially crosses human BMEC and HUVEC and that the human BMEC form a barrier to traversal. During the transmigration by the spirochetes, it was found that the integrity of the endothelial cell monolayers was maintained, as assessed by transendothelial electrical resistance measurements at the end of the experimental period, and thatB. burgdorferiappeared to bind human BMEC by their tips near or at cell borders, suggesting a paracellular route of transmigration. Importantly, traversal ofB. burgdorferiacross human BMEC induces the expression of plasminogen activators, plasminogen activator receptors, and matrix metalloproteinases. Thus, the fibrinolytic system linked by an activation cascade may lead to focal and transient degradation of tight junction proteins that allowsB. burgdorferito invade the CNS.
Sarcocystis neuronaMerozoites Express a Family of Immunogenic Surface Antigens That Are Orthologues of theToxoplasma gondiiSurface Antigens (SAGs) and SAG-Related Sequences
Authors: Howe Daniel K. , Gaji Rajshekhar Y. , Mroz-Barrett Meaghan , Gubbels Marc-Jan , Striepen Boris , Stamper Shelby
Published: January 2005

Sarcocystis neuronais a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of anS. neuronaexpressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences ofToxoplasma gondii. The primary peptide sequences of theS. neuronaproteins are consistent with the two-domain structure that has been described for theT. gondiiSAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface ofS. neuronamerozoites. Due to their surface localization and homology toT. gondiisurface antigens, theseS. neuronaproteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether theS. neuronaSAG family is as extensive as theT. gondiiSAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as moreS. neuronaESTs are generated. The existence of an SnSAG family inS. neuronaindicates that expression of multiple related surface antigens is not unique to the ubiquitous organismT. gondii. Instead, theSAGgene family is a common trait that presumably has an essential, conserved function(s).
Temporal Expression of Enteropathogenic
Escherichia coli
Virulence Genes in an In Vitro Model of Infection
Authors: Leverton Laura Q. , Kaper James B.
Published: January 2005

The hallmark of enteropathogenic
Escherichia coli
(EPEC) infection is the ability of EPEC to cause attaching and effacing (A/E) lesions on intestinal epithelium. This event is reproducible in in vitro tissue culture models of infection. We used real-time PCR to measure transcription from several locus of enterocyte effacement (LEE) operons (LEE1 to LEE5) and from
bfp
during a 5-h infection of HEp-2 cells with EPEC. We found that after the initial formation of A/E lesions, which occurs as early as 5 min postinfection, EPEC continues to increase transcription from LEE3 to LEE5 as well as from
bfp
. These levels are maximized by 3 h postinfection and remain constant throughout the course of infection. This increase in transcription from LEE3 to LEE5 occurs when LEE1 (
ler
) transcription is decreasing. EspA, EspB, intimin, Tir, and bundle-forming pilus expression is detectable during the entire 5-h infection. These results indicate that the EPEC genes involved in localized and intimate adherence are continually expressed after the initial stages of A/E lesion formation on host cells.
Expression of Major Histocompatibility Complex Class II and CD80 by Gingival Epithelial Cells Induces Activation of CD4+T Cells in Response to Bacterial Challenge
Authors: Matsuyama Takashi , Kawai Toshihisa , Izumi Yuichi , Taubman Martin A
Published: January 2005

HLA-DR (major histocompatibility complex [MHC] class II) is often expressed by epithelial cells in gingival tissues with periodontal disease but not by cells in healthy gingival tissues. Confocal microscopic analyses revealed that gingival epithelial cells (GEC) from tissue with periodontal disease express both HLA-DR and B7-1 (CD80) costimulatory molecules. Rat GEC lines were established to elucidate the possible role of MHC class II and B7-1 expression by GEC. Stimulation of a rat GEC line with gamma interferon (IFN-γ) induced the expression of MHC class II, whereas the cell line constitutively expressed B7-1 costimulatory molecules as determined by reverse transcription-PCR and flow cytometry.Actinobacillus actinomycetemcomitansOmp29-specific CD4+Th1 clone cells proliferated in response to pretreatment of GEC with fixedA. actinomycetemcomitansand IFN-γ. However, the Th1 cells did not respond to pretreatment of GEC with the bacteria alone or IFN-γ alone. The activation of Th1 clone cells induced by the GEC was inhibited by antibody to MHC class II or by CTLA4 immunoglobulin (CTLA4-Ig). Lymph node T cells did not demonstrate superantigen activity toA. actinomycetemcomitans, although both lymph node T cells and Th1 clone cells were sensitive to superantigen activity of staphylococcal enterotoxin A as cultured in the presence of IFN-γ-treated GEC. These results suggested that GEC can take up bacterial antigen and consequently process and present the bacterial antigen to CD4+T cells by MHC class II in conjunction with B7 costimulation. GEC appeared to play a role in the adaptive immune response by stimulating antigen-specific CD4+T cells.