bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibiotic-Sensitive TolC Mutants and Their Suppressors
Authors: Augustus Anne Marie , Celaya Teresa , Husain Fasahath , Humbard Matthew , Misra Rajeev
Published: March 2004

The TolC protein of
Escherichia coli
, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently,
tolC
null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of
tolC
missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic α-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex
+
phenotype. These novel phenotypes of TolC
R367H
and TolC
R390C
were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC
R367H
mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC
R367H
, mapping in the regulatory region of
acrAB
, uncoupled the AcrR-mediated repression of the
acrAB
genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal
acrR
gene was deleted or the
acrAB
genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.
Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite
Authors: Song Lingyun , Caguiat Jonathan , Li Zhongrui , Shokes Jacob , Scott Robert A. , Olliff Lynda , Summers Anne O.
Published: March 2004

The
repressor-activator MerR that controls transcription of the mercury
resistance (
mer
) operon is unusual for its high sensitivity
and specificity for Hg(II) in in vivo and in vitro transcriptional
assays. The metal-recognition domain of MerR resides at the homodimer
interface in a novel antiparallel arrangement of α-helix 5 that
forms a coiled-coil motif. To facilitate the study of this novel metal
binding motif, we assembled this antiparallel coiled coil into a single
chain by directly fusing two copies of the 48-residue α-helix 5
of MerR. The resulting 107-residue polypeptide, called the metal
binding domain (MBD), and wild-type MerR were overproduced and
purified, and their metal-binding properties were determined in vivo
and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair
of binding sites, just as MerR does, and it showed only a slightly
lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine
structure data showed that MBD has essentially the same Hg(II)
coordination environment as MerR. In vivo, cells overexpressing MBD
accumulated 70 to 100% more
203
Hg(II) than cells
bearing the vector alone, without deleterious effects on cell growth.
Both MerR and MBD variously bound other thiophilic metal ions,
including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We
conclude that (i) it is possible to simulate in a single polypeptide
chain the in vitro and in vivo metal-binding ability of dimeric,
full-length MerR and (ii) MerR's specificity in transcriptional
activation does not reside solely in the metal-binding
step.
DNA Interaction and Phosphotransfer of the C
4
-Dicarboxylate- Responsive DcuS-DcuR Two-Component Regulatory System from
Escherichia coli
Authors: Abo-Amer Aly E. , Munn Jonathan , Jackson Kerry , Aktas Murat , Golby Paul , Kelly David J. , Andrews Simon C.
Published: March 2004

The DcuS-DcuR system of
Escherichia coli
is a two-component sensor-regulator that controls gene expression in response to external C
4
-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C
4
-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [γ-
32
P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C
4
-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (
dctA
,
dcuB
, and
frdA
), with apparent

K
D

s of 6 to 32 μM for untreated DcuR and ≤1 to 2 μM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the
dcuB
,
dctA
, and
frdA
genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for
dcuB
that responds to DcuR.
Deletion of a Cyclic AMP Receptor Protein Homologue Diminishes Germination and Affects Morphological Development of
Streptomyces coelicolor
Authors: Derouaux A. , Halici S. , Nothaft H. , Neutelings T. , Moutzourelis G. , Dusart J. , Titgemeyer F. , Rigali S.
Published: March 2004

Open reading frame SCO3571 of
Streptomyces coelicolor
encodes a protein of the cyclic AMP (cAMP) receptor protein (CRP) superfamily of regulatory proteins. A mutant revealed a dramatic defect in germination, followed by growth delay and earlier sporulation. This phenotype correlates with those of an adenylate cyclase (
cya
) mutant that cannot synthesize cAMP. This finding suggests that
S. coelicolor
may use a Cya-cAMP-CRP system to trigger complex physiological processes such as morphogenesis.
Cellular XylS Levels Are a Function of Transcription of
xylS
from Two Independent Promoters and the Differential Efficiency of Translation of the Two mRNAs
Authors: González-Pérez M.-Mar , Ramos Juan L. , Marqués Silvia
Published: March 2004

XylS controls the expression of the
meta
-cleavage pathway for the metabolism of benzoates in
Pseudomonas putida
KT2440. The
xylS
gene is expressed from two promoters, Ps1 and Ps2. Transcription from Ps2 is low and constitutive, whereas transcription from Ps1 is induced in the presence of toluene. In this study, we also show that translation of mRNA generated from Ps1 is 10 times more efficient than that generated from Ps2. This pattern of transcription and translation of
xylS
gives rise to two modes of activation of the promoter of the
meta
pathway operon (Pm) according to the concentration of XylS in the cell. In cells growing with benzoate, with small amounts of XylS, the activated XylS regulator binds the effector and stimulates transcription from Pm, whereas in cells growing with toluene, the high levels of XylS suffice to stimulate transcription from Pm even in the absence of XylS effectors.
Downregulation of cGMP-dependent protein kinase expression by inflammatory cytokines in vascular smooth muscle cells
Authors: Browner Natasha C. , Sellak Hassan , Lincoln Thomas M.
Published: March 2004

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-Iβ, tumor necrosis factor (TNF)-α, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-Iβ, TNF-α, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor ( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5′-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5′-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC.
RyR1 exhibits lower gain of CICR activity than RyR3 in the SR: evidence for selective stabilization of RyR1 channel
Authors: Murayama Takashi , Ogawa Yasuo
Published: March 2004

We showed that frog α-ryanodine receptor (α-RyR) had a lower gain of Ca2+-induced Ca2+ release (CICR) activity than β-RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective “stabilization” of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953–2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [3H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [3H]ryanodine binding (B) was normalized by the number of maximal binding sites (Bmax), whereby the specific activity of each isoform was expressed. This B/Bmax expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca2+, Mg2+, adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog α-RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [3H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms.
Identification and characterization of a Kidd antigen/UT-B urea transporter expressed in human colon
Authors: Inoue Hideki , Jackson Shelley D. , Vikulina Tatyana , Klein Janet D. , Tomita Kimio , Bagnasco Serena M.
Published: March 2004

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kidd antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of ∼50- and ∼98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of 14C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa.
Phosphate uptake and PiT-1 protein expression in rat skeletal muscle
Authors: Abraham Kirk A. , Brault Jeffrey J. , Terjung Ronald L.
Published: March 2004

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (Pi) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of Pi into the cell must equal the rate of efflux, which is expected to be a function of intracellular Pi concentration. We measured 32P-labeled Pi uptake rates in different muscle fiber types to determine whether they are proportional to cellular Pi content. Pi uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 μmol·g−1·h−1), lower in red gastrocnemius (1.31 ± 0.11 μmol·g−1·h−1), and lowest in white gastrocnemius (0.49 ± 0.06 μmol·g−1·h−1). Reasonably similar rates were obtained in vivo. Pi uptake rates at plasma Pi concentrations of 0.3–1.7 mM confirm that the Pi uptake process is nearly saturated at normal plasma Pi levels. Pi uptake rate correlated with cellular Pi content ( r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of Pi uptake across fiber types implies that other factors are involved in regulating Pi uptake in skeletal muscle. Furthermore, fractional turnover of the cellular Pi pool (0.67, 0.57, and 0.33 h−1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular Pi, likely due to differences in resistance to Pi efflux from the fiber.
Troponin C isoform composition determines differences in Sr2+-activation characteristics between rat diaphragm fibers
Authors: O'Connell Brett , Stephenson D. George , Blazev Ronnie , Stephenson Gabriela M. M.
Published: March 2004

Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr2+ of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr2+ concentration ([Sr2+]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr2+ dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr2+ than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (−log10 [Sr2+]) curves is tightly ( r2 = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr2+ activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr2+, TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers.
θ-Isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-θ
Authors: Banan A. , Zhang L.J. , Shaikh M. , Fields J.Z. , Farhadi A. , Keshavarzian A.
Published: March 2004

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the θ-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-θ is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-θ (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-θ showed monolayer injury as indicated by decreased native PKC-θ activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-θ was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-θ was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-θ/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-θ led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-θ led to a mostly cytosolic distribution of θ-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC-θ isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC-θ involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-θ.
Copper overload affects copper and iron metabolism in Hep-G2 cells
Authors: Arredondo M. , Cambiazo V. , Tapia L. , González-Agüero M. , Núñez M. T. , Uauy R. , González M.
Published: March 2004

Divalent metal transporter #1 (DMT1) is responsible for intestinal nonheme Fe apical uptake. However, DMT1 appears to have an additional function in Cu transport in intestinal cells. Because the liver has an essential role in body Cu homeostasis, we examined the potential involvement of Cu in the regulation of DMT1 expression and activity in Hep-G2 cells. Cells exposed to 10 μM Cu exhibited a 22-fold increase in Cu content and a twofold decrease in Fe content compared with cells maintained in 0.4 μM Cu.64Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in Kmcompared with cells grown in 10 μM Cu. The decreased Kmmay represent an adaptive response to Cu deficiency. Cells treated with >50 μM Cu, showed an eightfold increase in cytosolic metallothionein. DMT1 protein decreased (35%), suggesting that intracellular Cu caused a reduction of DMT1 protein levels. Our data indicate that, as a result of Cu overload, Hep-G2 cells reduced their Fe content and their DMT1 protein levels. These findings strongly suggest a relationship between Cu and Fe homeostasis in Hep-G2 cells in which Cu accumulation downregulates DMT1 activity.
Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin
Authors: Lesnefsky Edward J. , Chen Qun , Slabe Thomas J. , Stoll Maria S. K. , Minkler Paul E. , Hassan Medhat O. , Tandler Bernard , Hoppel Charles L.
Published: March 2004

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.
Regulation of α7-integrin expression in vascular smooth muscle by injury-induced atherosclerosis
Authors: Chao Jun-Tzu , Meininger Gerald A. , Patterson Jan L. , Jones Sarah A. L. , Partridge Charles R. , Neiger Jessemy D. , Williams E. Spencer , Kaufman Stephen J. , Ramos Kenneth S. , Wilson Emily
Published: March 2004

Injury of vascular smooth muscle cells (VSMCs) by allylamine (AAM) leads to phenotypic changes associated with atherogenic progression including increased proliferation, migration, and alterations in cell adhesion. In the present study, the relationship between AAM-induced vascular injury and expression of the α7-integrin subunit was investigated. The α7-mRNA and protein expression were examined using real-time RT-PCR, fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and immunoblotting. In cultured VSMCs from aortas of AAM-treated rats (70 mg/kg for 20 days), α7-mRNA levels were increased more than twofold compared with control cells. No change was seen in β1-integrin expression. FACS analysis revealed increased cell surface expression of α7-protein (25 ± 9%; * P < 0.05). AAM treatment of naive VSMCs enhanced α7-mRNA expression (2.4 ± 0.7-fold, mean ± SE; * P < 0.05). The increased α7-mRNA expression was attenuated by the amine oxidase inhibitor semicarbazide and the antioxidant pyrrolidine dithiocarbamate, which confirms a role for oxidative stress in modulating α7-expression. In vivo α7-mRNA and protein expression were enhanced in the aortas of AAM-treated rats. In addition, increased α7-integrin expression facilitated AAM VSMC adhesion to laminin more efficiently compared with control (51 ± 2%; * P < 0.05). Chemical injury induced by AAM significantly enhances α7-integrin expression in VSMCs. These findings implicate for the first time the expression of α7-integrin during the response of VSMCs to vascular injury.
Prolonged tissue Po2 reduction after contraction in spinotrapezius muscle of spontaneously hypertensive rats
Authors: Smith Lane M. , Barbee R. Wayne , Ward Kevin R. , Pittman Roland N.
Published: March 2004

We tested the hypothesis that a deficit in oxygen extraction or an increase in oxygen demand after skeletal muscle contraction leads to delayed recovery of tissue oxygen tension (Po2) in the skeletal muscle of hypertensive rats compared with normotensive rats. Blood flow and Po2 recovery at various sites in the spinotrapezius muscle of spontaneously hypertensive rats (SHRs) were evaluated after a 3-min period of muscle contraction and were compared with corresponding values in Wistar-Kyoto rats (WKYs). The recovery of tissue Po2 [75 ± 7 (SHRs) vs. 99 ± 12% (WKYs) of resting values] and venular Po2 [72 ± 13 (SHRs) vs. 104 ± 10% (WKYs) of resting values] were significantly depressed in the SHRs 30 s postcontraction. The delayed recovery persisted for 120 s postcontraction for both tissue [86 ± 11 (SHRs) vs. 119 ± 13% (WKYs) of resting values] and venular [74 ± 2 (SHRs) vs. 100 ± 9% (WKYs) of resting values] Po2 levels. There was no significant difference in the recovery of arteriolar Po2 between the two groups 30 s postcontraction [95 ± 7 (SHRs) vs. 84 ± 8% (WKYs) of resting values]. Values for resting diameter of arcade arterioles in the two groups were not different [52 ± 3 (SHRs) vs. 51 ± 3 μm (WKYs)], but the arteriolar diameter after the 3-min contraction period was greater in the SHRs (71 ± 4 μm) than the WKYs (66 ± 4). Likewise, red blood cell (RBC) velocity [5.8 ± 0.3 (SHRs) vs. 4.7 ± 0.2 mm/s (WKYs)] and blood flow [23.0 ± 0.8 (SHRs) vs. 16.0 ± 1.0 nl/s (WKYs)] measurements were significantly greater in the SHRs at 30 s postcontraction. The delayed recovery of tissue Po2 in the SHRs compared with the WKYs can be explained by a decrease in oxygen diffusion from the rarefied microvascular network due to the increased RBC velocity and the shorter residence time in the microcirculation and the consequent disequilibrium for oxygen between plasma and RBCs. The delayed recovery of venular Po2 in the SHRs is consistent with this explanation, as venular Po2 is slowly restored to baseline by release of oxygen from the RBCs. This leaves the arterioles in the primary role as oxygen suppliers to restore Po2 in the tissue after muscle contraction.
Cav3.2 subunit underlies the functional T-type Ca2+ channel in murine hearts during the embryonic period
Authors: Niwa Noriko , Yasui Kenji , Opthof Tobias , Takemura Haruki , Shimizu Atsuya , Horiba Mitsuru , Lee Jong-Kook , Honjo Haruo , Kamiya Kaichiro , Kodama Itsuo
Published: March 2004

T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remodeling. Two voltage-gated Ca2+ subtypes (Cav3.1 and Cav3.2) have been cloned for the pore-forming α1-subunit of the T-type Ca2+ channel in cardiac muscle, but their differential roles remain to be clarified. The aim of this study was to elucidate the relative contribution of the two subtypes in the normal development of mouse hearts. A whole cell patch clamp was used to record ionic currents from ventricular myocytes isolated from mice of early (E9.5) and late embryonic days (E18) and from adult 10-wk-old mice. Large T-type Ca2+ current ( ICa,T) was observed at both E9.5 and E18, displaying similar voltage-dependence and kinetics of activation and inactivation. The current was inhibited by Ni2+ at relatively low concentrations (IC50 26–31 μM). ICa,T was undetectable in adult myocytes. Quantitative PCR analysis revealed that Cav3.2 mRNA is the predominant subtype encoding T-type Ca2+ channels at both E9.5 and E18. Cav3.1 mRNA increased from E9.5 to E18, but remained low compared with Cav3.2 mRNA during the whole embryonic period. In the adulthood, in contrast, Cav3.1 mRNA is greater than Cav3.2 mRNA. These results indicate that Cav3.2 underlies the functional T-type Ca2+ channels in the embryonic murine heart, and there is a subtype switching of transcripts from Cav3.2 to Cav3.1 in the perinatal period.
Subcutaneous obesity is not associated with sympathetic neural activation
Authors: Alvarez Guy E. , Ballard Tasha P. , Beske Stacy D. , Davy Kevin P.
Published: March 2004

We tested the hypothesis that muscle sympathetic nerve activity (MSNA) would not differ in subcutaneously obese (SUBOB) and nonobese (NO) men with similar levels of abdominal visceral fat despite higher plasma leptin concentrations in the former. We further hypothesized that abdominal visceral fat would be the strongest body composition- or regional fat distribution-related correlate of MSNA among these individuals. To accomplish this, we measured MSNA (via microneurography), body composition (via dual-energy X-ray absorptiometry), and abdominal fat distribution (via computed tomography) in 15 NO (body mass index ≤ 25 kg/m2; 22.4 ± 1.4 yr) and 9 SUBOB (25 ≤ body mass index ≤ 35 kg/m2; 23.4 ± 2.1 yr) sedentary men. As expected, body mass (94 ± 4 vs. 71 ± 2 kg), total fat mass (25 ± 2 vs. 12 ± 1 kg), and abdominal subcutaneous fat (307 ± 36 vs. 132 ± 12 cm2) were significantly higher in the SUBOB group compared with NO peers. However, the level of abdominal visceral fat did not differ significantly in the two groups (69 ± 7 vs. 55 ± 5 cm2). MSNA was not different between SUBOB and NO men (23 ± 3 vs. 24 ± 2 bursts/min; P > 0.05, respectively) despite ∼2.6-fold higher ( P < 0.05) plasma leptin concentration in the SUBOB men. Furthermore, abdominal visceral fat was the only body composition- or regional fat distribution-related correlate ( r = 0.45; P < 0.05) of MSNA in the pooled sample. In addition, abdominal visceral fat was related to MSNA in NO ( r = 0.58; P = 0.0239) but not SUBOB ( r = 0.39; P = 0.3027) men. Taken together with our previous observations, our findings suggest that the relation between obesity and MSNA is phenotype dependent. The relation between abdominal visceral fat and MSNA was evident in NO but not in SUBOB men and at levels of abdominal visceral fat below the level typically associated with elevated cardiovascular and metabolic disease risk. Our observations do not support an obvious role for leptin in contributing to sympathetic neural activation in human obesity and, in turn, are inconsistent with the concept of selective leptin resistance.
Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells
Authors: Sasamori Kan , Sasaki Tsukasa , Takasawa Shin , Tamada Tsutomu , Nara Masayuki , Irokawa Toshiya , Shimura Sanae , Shirato Kunio , Hattori Toshio
Published: March 2004

Cyclic ADP-ribose (cADPR), a putative Ca2+-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca2+release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 μM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca2+. Infusions of lower concentrations (2–4 μM) of cADPR or inositol 1,4,5-trisphosphate (IP3) alone were without effect on the baseline current, but a combined application of cADPR and IP3mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca2+in response to both IP3and cADPR. cADPR released Ca2+from microsomes desensitized to IP3or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP3, operates in airway gland acinar cells to mobilize Ca2+, resulting in Cl−secretion.
Compensation for an increase in body fat caused by donor transplants into mice
Authors: Rooks Cherie , Bennet TaNeisha , Bartness Timothy J. , Harris Ruth B. S.
Published: March 2004

Rodents tend to compensate for experimental obesity in which both adipocyte size and number are increased. In contrast, it was recently reported that Siberian hamsters do not compensate for dorsal subcutaneous transplants of fat, which increase body fat without changing the size of adipocytes. In the first experiment described here we tested whether mice changed the size of their endogenous fat stores 2 or 5 wk after donor fat was added as subcutaneous transplants. Each epididymal fat pad from donor mice was cut in half and placed ventrally in recipient mice, increasing body fat by ∼10%. After 2 wk, there was no effect of the transplants on the size of endogenous fat depots or the size of adipocytes in epididymal fat depots. There was a substantial decrease in mass and adipocyte size in transplanted fat. Five weeks after surgery the endogenous epididymal and retroperitoneal fat depots of recipient mice were significantly decreased, serum leptin was reduced, and the size of adipocytes in endogenous epididymal fat was significantly reduced, although cell number had not changed. The size of transplanted cells was the same as at 2 wk. In a second experiment, epididymal fat was placed as either dorsal or ventral subcutaneous fat transplants. Five weeks after surgery the endogenous fat depots were decreased in all recipient mice but none of the differences reached statistical significance. These results suggest that mice have mechanisms to maintain total body fat mass that respond to an increase in the number of fat cells present.
Maternal LPS induces cytokines in the amniotic fluid and corticotropin releasing hormone in the fetal rat brain
Authors: Gayle Dave A. , Beloosesky Ron , Desai Mina , Amidi Fataneh , Nuñez Sonia E. , Ross Michael G.
Published: March 2004

Perinatal infections are a risk factor for fetal neurological pathologies, including cerebral palsy and schizophrenia. Cytokines that are produced as part of the inflammatory response are proposed to partially mediate the neurological injury. This study investigated the effects of intraperitoneal injections of lipopolysaccharide (LPS) to pregnant rats on the production of cytokines and stress markers in the fetal environment. Gestation day 18 pregnant rats were treated with LPS (100 μg/kg body wt ip), and maternal serum, amniotic fluid, placenta, chorioamnion, and fetal brain were harvested at 1, 6, 12, and 24 h posttreatment to assay for LPS-induced changes in cytokine protein (ELISA) and mRNA (real-time RT-PCR) levels. We observed induction of proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) as well as the anti-inflammatory cytokine IL-10 in the maternal serum within 6 h of LPS exposure. Similarly, proinflammatory cytokines were induced in the amniotic fluid in response to LPS; however, no significant induction of IL-10 was observed in the amniotic fluid. LPS-induced mRNA changes included upregulation of the stress-related peptide corticotropin-releasing factor in the fetal whole brain, TNF-α, IL-6, and IL-10 in the chorioamnion, and TNF-α, IL-1β, and IL-6 in the placenta. These findings suggest that maternal infections may lead to an unbalanced inflammatory reaction in the fetal environment that activates the fetal stress axis.
Sodium transport antagonism reduces thrombotic microangiopathy in stroke-prone spontaneously hypertensive rats
Authors: Sepehrdad Reza , Chander Praveen N. , Singh Gagan , Stier Charles T.
Published: March 2004

We examined whether amiloride, an agent that possesses epithelial sodium channel (ENaC)- and sodium/hydrogen exchange (NHE)-inhibitory activities, would exhibit renal vascular protection in saline-drinking, stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP received amiloride (1.0 mg·kg−1·day−1, n = 6) or deionized water (3 mg·kg−1·day−1, n = 6) for 5 wk starting at 61 days of age. Systolic blood pressure (SBP) did not differ among the groups, and there was no difference in the average daily urine output, sodium excretion, or potassium excretion. Terminal urinary protein excretion, blood urea nitrogen, and renal thrombotic microangiopathic lesions were markedly reduced in the amiloride group with no difference in plasma renin activity (PRA). In a survival protocol, SHRSP infused subcutaneously with benzamil (0.7 mg·kg−1·day−1, n = 8), a selective ENaC inhibitor, dimethylamiloride (0.7 mg·kg−1·day−1, n = 8), a selective NHE inhibitor, or vehicle ( n = 7) had comparable SBP. Dimethylamiloride nonetheless prolonged survival of SHRSP ( P < 0.005 vs. vehicle), and benzamil-treated SHRSP lived even longer ( P < 0.0001 vs. vehicle; P < 0.05 vs. dimethylamiloride). In a separate series, plasma potassium concentration was elevated by dimethylamiloride (3.4 ± 0.1 meq/l, n = 8) and benzamil (3.3 ± 0.1 meq/l, n = 8) relative to vehicle (3.0 ± 0.1 meq/l, n = 8) at 4 but not at 24 h after dosing. These findings suggest the involvement of a sodium transport mechanism in the development of thrombotic microangiopathy in SHRSP, unrelated to marked changes in arterial pressure, PRA, plasma potassium, or urinary water and electrolyte excretion.
Altered expression of urea transporters in response to ureteral obstruction
Authors: Li Chunling , Klein Janet D. , Wang Weidong , Knepper Mark A. , Nielsen Søren , Sands Jeff M. , Frøkiær Jørgen
Published: March 2004

Urea plays an important role in the urinary concentrating capacity. Renal inner medullary (IM) urea transporter expression was examined in rats with bilateral (BUO) or unilateral ureteral obstruction (UUO). BUO (24 h) was associated with markedly increased plasma urea (42.4 ± 1.0 vs. 5.2 ± 0.2 mmol/l) and a significant decease in expression of UT-A1 (28 ± 8% of sham levels), UT-A3 (45 ± 11%), and UT-B (70 ± 8%). Immunocytochemistry confirmed downregulation of UT-A1 and UT-A3 in IM collecting duct and UT-B in the descending vasa recta. Three days after release of BUO, UT-A1, UT-A3, and UT-B remained significantly downregulated (UT-A1: 37 ± 6%; UT-A3: 25 ± 6%; and UT-B: 10 ± 5% of sham levels; P < 0.05) concurrent with a persistent polyuria and a marked reduction in solute-free water reabsorption (115 ± 11 vs. 196 ± 8 μl·min−1·kg−1, P < 0.05). Moreover, 14 days after release of BUO, total UT-A1, UT-A3, and UT-B remained significantly decreased compared with sham-operated controls and urine urea remained reduced (588 ± 43 vs. 1,150 ± 94 mmol/l). Consistent with increased levels of plasma urea 24 h after onset of UUO (7.4 ± 0.3 vs. 4.8 ± 0.3 mmol/l), the protein abundance of UT-A1, UT-A3, and UT-B in IM was markedly reduced in the obstructed kidney, which was confirmed by immunocytochemistry. In the nonobstructed kidney, the expression of urea transporters did not change. In conclusion, reduced expression of UT-A1, UT-A3, and UT-B levels in both BUO and UUO rats suggests that urea transporters play important roles in the impaired urinary concentrating capacity in response to urinary tract obstruction.
Effect of ventilation rate on instilled surfactant distribution in the pulmonary airways of rats
Authors: Anderson Joseph C. , Molthen Robert C. , Dawson Christopher A. , Haworth Steve T. , Bull Joseph L. , Glucksberg Matthew R. , Grotberg James B.
Published: March 2004

Liquid can be instilled into the pulmonary airways during medical procedures such as surfactant replacement therapy, partial liquid ventilation, and pulmonary drug delivery. For all cases, understanding the dynamics of liquid distribution in the lung will increase the efficacy of treatment. A recently developed imaging technique for the study of real-time liquid transport dynamics in the pulmonary airways was used to investigate the effect of respiratory rate on the distribution of an instilled liquid, surfactant, in a rat lung. Twelve excised rat lungs were suspended vertically, and a single bolus (0.05 ml) of exogenous surfactant (Survanta, Ross Laboratories, Columbus, OH) mixed with radiopaque tracer was instilled as a plug into the trachea. The lungs were ventilated with a 4-ml tidal volume for 20 breaths at one of two respiratory rates: 20 or 60 breaths/min. The motion of radiodense surfactant was imaged at 30 frames/s with a microfocal X-ray source and an image intensifier. Dynamics of surfactant distribution were quantified for each image by use of distribution statistics and a homogeneity index. We found that the liquid distribution depended on the time to liquid plug rupture, which depends on ventilation rate. At 20 breaths/min, liquid was localized in the gravity-dependent region of the lung. At 60 breaths/min, the liquid coated the airways, providing a more vertically uniform liquid distribution.
Fast force-generation dynamics of human articulatory muscles
Authors: Ito Takayuki , Murano Emi Z. , Gomi Hiroaki
Published: March 2004

To explore the mechanisms of speech articulation, which is one of the most sophisticated human motor skills controlled by the central nervous system, we investigated the force-generation dynamics of the human speech articulator muscles [orbicularis oris superior (OOS) and inferior (OOI) muscles of the lips]. Short-pulse electrical stimulation (300 μs) with approximately three or four times the sensation threshold intensity of each subject induced the muscle response. The responses of these muscles were modeled as second-order dynamics with a time delay (TD), and the model parameters [natural frequency (NF), damping ratio (DR), and TD] were identified with a nonlinear least mean squares method. The OOS (NF: 6.1 Hz, DR: 0.71, TD: 14.5 ms) and OOI (NF: 6.1 Hz, DR: 0.68, TD: 15.6 ms) showed roughly similar characteristics in eight subjects. The dynamics in the tongue (generated by combined muscles) also showed similar characteristics (NF: 6.1 Hz, DR: 0.68, TD: 17.4 ms) in two subjects. The NF was higher, and the DR was lower than results measured for arm muscles (NF: 4.25 Hz, DR: 1.05, TD: 23.8 ms for triceps long head), indicating that articulatory organs adapt for more rapid movement. In contrast, slower response dynamics was estimated when muscle force data by voluntarily contraction task were used for force-generation dynamics modeling. We discuss methodological problems in estimating muscle dynamics when different kinds of muscle contraction methods are used.
Measurement of inferior vena cava diameter for evaluation of venous return in subjects onday 10of a bed-rest experiment
Authors: Ishizaki Yuko , Fukuoka Hideoki , Ishizaki Tatsuro , Kino Minoru , Higashino Hirohiko , Ueda Nobuo , Fujii Yuri , Kobayashi Yohnosuke
Published: March 2004

We evaluated the usefulness of measurements of the inferior vena cava (IVC) diameters on abdominal echograms as an indicator of changes of venous return in subjects with orthostatic intolerance (OI) induced by simulated microgravity. We performed a standing test and recorded the IVC diameters on abdominal echograms in 12 subjects placed on a 20-day 6° head-down-tilt bed-rest experiment. We found that different patterns of changes in IVC diameter occurred in the standing test on day 10 of the experiment; in five subjects with a marginal decrease in pulse pressure, IVC diameters in the upright position were markedly decreased compared with those in the supine position. In five subjects with feelings of discomfort, the IVC diameters in the upright position distended or did not decrease from those in the supine position. These results suggested that the changes in IVC diameter on the standing test indicated the presence of various types of hemodynamic responses of OI caused by simulated microgravity. In this study, we also evaluated changes in body-water compartments by conducting multifrequency bioelectrical impedance analysis. Longitudinal data analysis showed that the total body-water-to-fat-free mass and extracellular fluid-to-fat-free mass ratios decreased during the experimental period and recovered thereafter, and that the ratio of intracellular fluid to fat-free mass decreased during the experiment. No significant difference in changes in body-water compartments was seen among subjects with different patterns of changes in IVC diameters. Measurement of IVC diameter was useful to estimate hemodynamic changes in subjects with OI.
Negative metabolic and coronary flow effects of decreases in cAMP and increases in cGMP in control and renal hypertensive rabbit hearts
Authors: Rodriguez Roberto , Molino Bruno , Weiss Harvey R. , Scholz Peter M.
Published: March 2004

The interaction during stimulation of cGMP and inhibition of cAMP was investigated in control and renal hypertensive hearts. Control and hypertensive [1 kidney, 1 clip (1K1C)] rabbits were used. The anesthetized open-chest groups were vehicle, 8-bromo-cGMP (8-Br-cGMP; 10−3M), propranolol (Prop; 2 mg/kg), and Prop + 8-Br-cGMP. O2 consumption levels (V̇o2) in the subepicardium (Epi) and subendocardium (Endo) were determined from coronary flow (microspheres) and O2 extraction (microspectrophotometry). Wall thickening and cAMP levels were also determined. In control, no significant change in V̇o2 was seen for the 8-Br-cGMP group, but V̇o2 was decreased from Epi (9.7 ± 1.5 ml O2·min−1·100 g−1) and Endo (10.5 ± 0.4 ml O2·min−1·100 g−1) to 6.8 ± 0.6/7.8 ± 0.5 ml O2·min−1·100 g−1 in the control Prop group. Control Prop + 8-Br-cGMP did not cause a further fall in V̇o2 but lowered Endo flow. In 1K1C, V̇o2 decreased from Epi/Endo (10.8 ± 1.3/11 ± 1.0 ml O2·min−1·100 g−1) to 7.8 ± 1.1/8.7 ± 0.5 ml O2·min−1·100 g−1 in the 1K1C 8-Br-cGMP group and to 7 ± 0.5/8.1 ± 0.5 ml O2·min−1·100 g−1 in the 1K1C Prop group. 1K1C Prop + 8-Br-cGMP did not cause a further fall in V̇o2 but lowered blood flow. No significant changes in cAMP levels were present with 8-Br-cGMP in control or 1K1C rabbits, but significant decreases were seen with Prop in both control and 1K1C rabbits. No further change was seen in Prop + 8-Br-cGMP for either control or 1K1C. Thus the negative metabolic effect of stimulating cGMP was seen only in the hypertensive rabbit heart. The negative metabolic effect of inhibiting cAMP was seen in both the control and the hypertensive rabbit heart. However, the negative metabolic effects of cGMP and cAMP were nonadditive.