bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuous mild heat stress induces differentiation of mammalian myoblasts, shifting fiber type from fast to slow
Authors: Yamaguchi Tetsuo , Suzuki Takayoshi , Arai Hideaki , Tanabe Shihori , Atomi Yoriko
Published: July 2009

Local hyperthermia has been widely used as physical therapy for a number of diseases such as inflammatory osteoarticular disorders, tendinitis, and muscle injury. Local hyperthermia is clinically applied to improve blood and lymphatic flow to decrease swelling of tissues (e.g., skeletal muscle). As for muscle repair following injury, the mechanisms underlying the beneficial effects of hyperthermia-induced muscle repair are unknown. In this study, we investigated the direct effects of continuous heat stress on the differentiation of cultured mammalian myoblasts. Compared with control cultures grown at 37°C, incubation at 39°C (continuous mild heat stress; CMHS) enhanced myotube diameter, whereas myotubes were poorly formed at 41°C by primary human skeletal muscle culture cells, human skeletal muscle myoblasts (HSMMs), and C2C12 mouse myoblasts. In HSMMs and C2C12 cells exposed to CMHS, mRNA and protein levels of myosin heavy chain (MyHC) type I were increased compared with the control cultures. The mRNA level of MyHC IIx was unaltered in HSMMs and decreased in C2C12 cells, compared with cells that were not exposed to heat stress. These results indicated a fast-to-slow fiber-type shift in myoblasts. We also examined upstream signals that might be responsible for the fast-to-slow shift of fiber types. CMHS enhanced the mRNA and protein levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α in HSMMS and C2C12 cells but not the activities of MAPKs (ERK1/2 and p38 MAPK) in HSMMs and C2C12 cells. These data suggest that CMHS induces a fast-to-slow fiber-type shift of mammalian myoblasts through PGC-1α.
Inhibition of basal p38 or JNK activity enhances epithelial barrier function through differential modulation of claudin expression
Authors: Carrozzino Fabio , Pugnale Paolo , Féraille Eric , Montesano Roberto
Published: July 2009

Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH2-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38α by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38β only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na+ and Cl− across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.
Malformed mdx myofibers have normal cytoskeletal architecture yet altered EC coupling and stress-induced Ca2+ signaling
Authors: Lovering Richard M. , Michaelson Luke , Ward Christopher W.
Published: July 2009

Skeletal muscle function is dependent on its highly regular structure. In studies of dystrophic ( dy/dy) mice, the proportion of malformed myofibers decreases after prolonged whole muscle stimulation, suggesting that the malformed myofibers are more prone to injury. The aim of this study was to assess morphology and to measure excitation-contraction (EC) coupling (Ca2+ transients) and susceptibility to osmotic stress (Ca2+ sparks) of enzymatically isolated muscle fibers of the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles from young (2–3 mo) and old (8–9 mo) mdx and age-matched control mice (C57BL10). In young mdx EDL, 6% of the myofibers had visible malformations (i.e., interfiber splitting, branched ends, midfiber appendages). In contrast, 65% of myofibers in old mdx EDL contained visible malformations. In the mdx FDB, malformation occurred in only 5% of young myofibers and 11% of old myofibers. Age-matched control mice did not display the altered morphology of mdx muscles. The membrane-associated and cytoplasmic cytoskeletal structures appeared normal in the malformed mdx myofibers. In mdx FDBs with significantly branched ends, an assessment of global, electrically evoked Ca2+ signals (indo-1PE-AM) revealed an EC coupling deficit in myofibers with significant branching. Interestingly, peak amplitude of electrically evoked Ca2+ release in the branch of the bifurcated mdx myofiber was significantly decreased compared with the trunk of the same myofiber. No alteration in the basal myoplasmic Ca2+ concentration (i.e., indo ratio) was seen in malformed vs. normal mdx myofibers. Finally, osmotic stress induced the occurrence of Ca2+ sparks to a greater extent in the malformed portions of myofibers, which is consistent with deficits in EC coupling control. In summary, our data show that aging mdx myofibers develop morphological malformations. These malformations are not associated with gross disruptions in cytoskeletal or t-tubule structure; however, alterations in myofiber Ca2+ signaling are evident.
Regulation of intracerebral arteriolar tone by Kv channels: effects of glucose and PKC
Authors: Straub Stephen V. , Girouard Helene , Doetsch Paul E. , Hannah Rachael M. , Wilkerson M. Keith , Nelson Mark T.
Published: July 2009

Voltage-gated potassium (Kv) channels in vascular smooth muscle cells (VSMC) are critical regulators of membrane potential and vascular tone. These channels exert a hyperpolarizing influence to counteract the depolarizing effects of intraluminal pressure and vasoconstrictors. However, the contribution of Kv channel activity to the functional regulation of cerebral (parenchymal) arterioles within the brain is not known. Thus Kv channel properties in parenchymal arteriolar SMCs were characterized. Isolated, pressurized parenchymal arterioles and arterioles in cortical brain slices exhibited robust constriction in the presence of the Kv channel inhibitor 4-aminopyridine (4-AP). 4-AP also decreased the amplitude of Kv currents recorded from SMCs. The steady-state activation and inactivation properties of Kv currents suggested that these channels are composed of Kv1.2 and 1.5 subunits, which was confirmed by RT-PCR. Kv channels can be regulated by extracellular glucose, which may be involved in the functional hyperemic response in the brain. Thus the effects of glucose on Kv channel activity and arteriolar function were investigated. Elevation of glucose from 4 to 14 mM significantly decreased the peak Kv current amplitude and constricted arterioles. Arteriolar constriction was prevented by inhibition of protein kinase C (PKC), consistent with previous studies showing enhanced PKC activity in the presence of elevated glucose. In cortical brain slices, the dilation generated by neuronal activity induced by electrical field stimulation was decreased by 54% in 14 mM glucose when compared with the dilation in 4 mM glucose. In anesthetized mice the whisker stimulation-induced increase in local cerebral blood flow was also significantly decreased in 14 mM glucose, and this effect was similarly prevented by PKC inhibition. These findings point to a critical role for Kv channels in the regulation of intracerebral arteriolar function and suggest that changes in perivascular glucose levels could directly alter vascular diameter resulting in a modulation of local cerebral blood flow.
PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells
Authors: Padovano Valeria , Massari Silvia , Mazzucchelli Silvia , Pietrini Grazia
Published: July 2009

Here we show that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of the excitatory amino acid carrier 1 (EAAC1) glutamate transporter from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells to intracellular compartments. The PKC-induced internalization of EAAC1 is negatively regulated by the calcineurin inhibitor cyclosporine A and by the expression of a dominant-negative mutant of the endocytic protein dynamin 1, a well-known target of the phosphatase activity of calcineurin. Using 32P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a target of these modifications was the serine778 residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin and that this activation is required to mediate internalization of EAAC1 in MDCK cells. By immunofluorescence experiments with endosomal markers we demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral betaine-GABA transporter BGT1 and activated PKCα. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a posttreatment with a PKC-specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together, our data indicate that PKC activity regulates EAAC1 surface density in MDCK cells by inducing its internalization and retention in PKCα-labeled recycling endosomes common to apical and basolateral proteins.
Characterization of an epilepsy-associated variant of the human Cl−/HCO3− exchanger AE3
Authors: Vilas Gonzalo L. , Johnson Danielle E. , Freund Paul , Casey Joseph R.
Published: July 2009

Anion exchanger 3 (AE3), expressed in the brain, heart, and retina, extrudes intracellular HCO3− in exchange for extracellular Cl−. The SLC4A3 gene encodes two variants of AE3, brain or full-length AE3 (AE3fl) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. The AE3-A867D allele in humans has been associated with the development of IGE (IGE), which accounts for ∼30% of all epilepsies. To examine the molecular basis for the association of the A867D allele with IGE, we characterized wild-type (WT) and AE3fl-A867D in transfected human embryonic kidney (HEK)-293 cells. AE3fl-A867D had significantly reduced transport activity relative to WT (54 ± 4%, P < 0.01). Differences in expression levels or the degree of protein trafficking to the plasma membrane did not account for the defect of AE3fl-A867D. Treatment with 8-bromo-cAMP (8-Br-cAMP) increased Cl−/HCO3− exchange activity of WT and AE3fl-A867D to a similar degree, which was abolished by preincubation with the protein kinase A (PKA)-specific inhibitor H89. This indicates that PKA regulates WT and AE3fl-A867D Cl−/HCO3− exchange activity. No difference in Cl−/HCO3− exchange activity was found between cultures of mixed populations of neonatal hippocampal cells from WT and slc4a3−/− mice. We conclude that the A867D allele is a functional (catalytic) mutant of AE3 and that the decreased activity of AE3fl-A867D may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexcitability and the generation of seizures.
Bile acids inhibit NAD+-dependent 15-hydroxyprostaglandin dehydrogenase transcription in colonocytes
Authors: Miyaki Akira , Yang Peiying , Tai Hsin-Hsiung , Subbaramaiah Kotha , Dannenberg Andrew J.
Published: July 2009

Multiple lines of evidence have suggested a role for both bile acids and prostaglandins (PG) in gastrointestinal carcinogenesis. Levels of PGE2 are determined by both synthesis and catabolism. Previously, bile acid-mediated induction of cyclooxygenase-2 (COX-2) was found to stimulate PGE2 synthesis. NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme responsible for the catabolism of PGE2, has been linked to colorectal carcinogenesis. In this study, we determined whether bile acids altered the expression of 15-PGDH in human colon cancer cell lines. Treatment with unconjugated bile acids (chenodeoxycholate and deoxycholate) suppressed the transcription of 15-PGDH, resulting in reduced amounts of 15-PGDH mRNA, protein, and enzyme activity. Conjugated bile acids were less potent suppressors of 15-PGDH expression than unconjugated bile acids. Treatment with chenodeoxycholate activated protein kinase C (PKC), leading in turn to increased extracellular signal-regulated kinase (ERK) 1/2 activity. Small molecules that inhibited bile acid-mediated activation of PKC and ERK1/2 also blocked the downregulation of 15-PGDH. Bile acids induced early growth response factor-1 (Egr-1) and Snail, a repressive transcription factor that bound to the 15-PGDH promoter. Silencing Egr-1 or Snail blocked chenodeoxycholate-mediated downregulation of 15-PGDH. Together, these data indicate that bile acids activate the signal transduction pathway PKC → ERK1/2 → Egr-1 → Snail and thereby suppress 15-PGDH transcription. Bile acids appear to increase the release of PGs from cells by downregulating catabolism in addition to stimulating synthesis. These results provide new mechanistic insights into the link between bile acids and gastrointestinal carcinogenesis.
Changes in intestinal Toll-like receptors and cytokines precede histological injury in a rat model of necrotizing enterocolitis
Authors: Liu Yuying , Zhu Limin , Fatheree Nicole Y. , Liu Xiaoqin , Pacheco Susan E. , Tatevian Nina , Rhoads Jon Marc
Published: July 2009

It is unclear whether the broad inflammatory response shown in neonatal necrotizing enterocolitis (NEC) is the cause or the effect of tissue injury. Toll-like receptors (TLRs) on intestinal dendritic, mononuclear, and epithelial cells recognize bacterial ligands and damaged tissues, thus activating the inflammatory response. The present study aimed to determine whether active TLR signaling would precede histological injury in NEC. Newborn rat pups were divided into four groups: dam fed, dam fed-hypoxic, formula fed, and formula fed-hypoxic (NEC). The ileal tissues were evaluated for NEC scores at 24, 48, 72, and 120 h. Quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure and localize intestinal TLRs. Cytokines were assessed by a multispot cytokine array. Among the four groups, ileal injury was seen only after 72 h of formula feeding and hypoxia. We found selective induction of mRNA levels in NEC compared with dam-fed controls for TLR2 > TLR4 > TLR1 = TLR3, TLR7, and TLR9 > TLR6 ( P < 0.01); TLR5 was downregulated ( P < 0.01). All TLR changes started at 48 h, before any histological evidence of NEC. Both Th1-type cytokines (IFN-γ, IL-1β, TNF-α, and KC/GRO) and Th2-type cytokines (IL-4, IL-5 and IL-13) were significantly increased in NEC but also in nondamaged formula-fed rat ileum. In conclusion, the intestinal expression of TLRs and cytokines precedes histological injury in the experimental NEC.
Cerebral oxygenation and metabolism during exercise following three months of endurance training in healthy overweight males
Authors: Seifert T. , Rasmussen P. , Brassard P. , Homann P. H. , Wissenberg M. , Nordby P. , Stallknecht B. , Secher N. H. , Nielsen H. B.
Published: July 2009

Endurance training improves muscular and cardiovascular fitness, but the effect on cerebral oxygenation and metabolism remains unknown. We hypothesized that 3 mo of endurance training would reduce cerebral carbohydrate uptake with maintained cerebral oxygenation during submaximal exercise. Healthy overweight males were included in a randomized, controlled study (training: n = 10; control: n = 7). Arterial and internal jugular venous catheterization was used to determine concentration differences for oxygen, glucose, and lactate across the brain and the oxygen-carbohydrate index [molar uptake of oxygen/(glucose + ½ lactate); OCI], changes in mitochondrial oxygen tension (ΔPMitoO2) and the cerebral metabolic rate of oxygen (CMRO2) were calculated. For all subjects, resting OCI was higher at the 3-mo follow-up (6.3 ± 1.3 compared with 4.7 ± 0.9 at baseline, mean ± SD; P < 0.05) and coincided with a lower plasma epinephrine concentration ( P < 0.05). Cerebral adaptations to endurance training manifested when exercising at 70% of maximal oxygen uptake (∼211 W). Before training, both OCI (3.9 ± 0.9) and ΔPMitoO2(−22 mmHg) decreased ( P < 0.05), whereas CMRO2increased by 79 ± 53 micromol·100·g−1min−1( P < 0.05). At the 3-mo follow-up, OCI (4.9 ± 1.0) and ΔPMitoO2(−7 ± 13 mmHg) did not decrease significantly from rest and when compared with values before training ( P < 0.05), CMRO2did not increase. This study demonstrates that endurance training attenuates the cerebral metabolic response to submaximal exercise, as reflected in a lower carbohydrate uptake and maintaind cerebral oxygenation.
Passive diffusion of naltrexone into human and animal cells and upregulation of cell proliferation
Authors: Cheng Fan , McLaughlin Patricia J. , Banks William A. , Zagon Ian S.
Published: July 2009

Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide, opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells, NTX was fluorescently labeled [1-( N)-fluoresceinyl NTX thiosemicarbazone; FNTX] to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line (SCC-1) was incubated with FNTX for as little as 1 min, cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopy, with enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2), African green monkey kidney cell line (COS-7), and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 4°C, with the signal being comparable to that recorded at 37°C. A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein-labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis, cells incubated with FNTX at concentrations ranging from 10−5to 10−8M had a 5-bromo-2′-deoxyuridine index that was 39–82% greater than for vehicle-treated cells and was comparable to that of unlabeled NTX (37–70%). Taken together, these results suggested that NTX enters cells by passive diffusion in a nonsaturable manner.
The JNK MAP kinase pathway contributes to the development of endotoxin-induced diaphragm caspase activation
Authors: Supinski Gerald S. , Ji Xinying , Callahan Leigh Ann
Published: July 2009

We previously demonstrated that endotoxin-induced sepsis results in caspase 8-mediated diaphragmatic dysfunction. The upstream signaling pathways modulating diaphragm caspase 8 activation in response to endotoxin administration are, however, unknown. The purpose of the present study was to test the hypothesis that the JNK (Jun N-terminal Kinase) pathway is activated in the diaphragm during sepsis and contributes to sepsis-induced diaphragm caspase 8 activation. Endotoxin was administered to intact animals to model the effects of sepsis. We first assessed the time course of JNK activation after endotoxin (12 mg/kg ip) administration to mice. We then determined whether JNK inhibitor administration ( 30 μm/kg ip SP600125) could prevent caspase 8 activation and diaphragm weakness in endotoxin-treated mice. Experiments were then repeated comparing the effects of endotoxin on control and transgenic JNK knockout mice. We finally determined whether cytomix (LPS, TNFα, IL1β, and IFN-γ) exposure activated caspase 8 in C2C12 muscle cells and whether caspase 8 activation was attenuated by either chemical inhibition of JNK (30 μM SP600125) or transfection with a dominant negative JNK construct. We found that endotoxin activated diaphragm JNK ( P < 0.001) and increased active caspase 8 ( P < 0.01). Inhibition of JNK with SP600125 or by use of JNK-deficient animals prevented diaphragm caspase 8 activation ( P < 0.01) and prevented diaphragm weakness ( P < 0.05). JNK inhibition also prevented caspase 8 activation in cytokine-treated muscle cells ( P < 0.001). These data implicate JNK activation as a major factor mediating inflammation-induced skeletal muscle caspase 8 activation and weakness.
EGFR-PLCγ1 signaling mediates high glucose-induced PKCβ1-Akt activation and collagen I upregulation in mesangial cells
Authors: Wu D. , Peng F. , Zhang B. , Ingram A. J. , Kelly D. J. , Gilbert R. E. , Gao B. , Kumar S. , Krepinsky J. C.
Published: July 2009

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We have recently shown that epidermal growth factor receptor (EGFR) transactivation mediates high glucose (HG)-induced collagen I upregulation through PI3K-PKCβ1-Akt signaling in mesangial cells (MC). Phospholipase Cγ1 (PLCγ1) interacts with activated growth factor receptors and activates classic PKC isoforms. We thus studied its role in HG-induced collagen I upregulation in MC. Primary rat MC were treated with HG (30 mM) or mannitol as osmotic control. Protein kinase activation was assessed by Western blotting and collagen I upregulation by Northern blotting. Diabetes was induced in rats by streptozotocin. HG treatment for 1 h led to PLCγ1 membrane translocation and Y783 phosphorylation, both indicative of its activation. Mannitol was without effect. PLCγ1 Y783 phosphorylation was also seen in cortex and glomeruli of diabetic rats. HG induced a physical association between EGFR and PLCγ1 as identified by coimmunoprecipitation. PLCγ1 activation required EGFR kinase activity since it was prevented by the EGFR inhibitor AG1478 or overexpression of kinase-inactive EGFR (K721A). Phosphoinositide-3-OH kinase inhibition also prevented PLCγ1 activation. HG-induced Akt S473 phosphorylation, effected by PKCβ1, was inhibited by the PLCγ inhibitor U73122. PLCγ1 inhibition or downregulation by small interference RNA also prevented HG-induced collagen I upregulation. Our results indicate that EGFR-PLCγ1 signaling mediates HG-induced PKCβ1-Akt activation and subsequent collagen I upregulation in MC. Inhibition of EGFR or PLCγ1 may provide attractive therapeutic targets for the treatment of diabetic nephropathy.
Impaired transcription factor interplay in addition to advanced glycation end products suppress podocalyxin expression in high glucose-treated human podocytes
Authors: Drossopoulou Garyfalia I. , Tsotakos Nikolaos E. , Tsilibary Effie C.
Published: July 2009

Podocalyxin represents a Wilms’ tumor suppressor protein (WT1)-regulated differentiation marker for glomerular epithelium. We provide evidence concerning mechanisms involved in the regulation of podocalyxin expression following long-term exposure to increased (25 mM) glucose levels. Prolonged culture of conditionally immortalized human podocytes in 25 mM glucose induced suppression of podocalyxin expression both at the protein and mRNA levels, whereas WT1 protein levels remained unaltered. WT1 interacted with another transcription factor, CRE-binding protein (CBP). This association was decreased by 40% in the presence of 25 mM glucose. Chromatin immunoprecipitation assays on chromatin from podocytes cultured in 25 mM glucose revealed reduced WT1 binding to podocalyxin promoter sequences, probably resulting from impaired WT1-CBP interactions. We explored the possible role of glucose-induced adducts (advanced glycation end products; AGEs) in impairing interactions between WT1 and CBP, with the use of aminoguanindine, an inhibitor of AGE formation. Podocytes were cultured in the simultaneous presence of 20 mM aminoguanidine and 25 mM glucose, and podocalyxin protein levels were examined. Aminoguanidine effectively prevented downregulation of podocalyxin protein levels but could not restore podocalyxin levels once expression was suppressed. Thus increased glucose apparently impaired the ability of WT1 to initiate transcription in part by decreased association of WT1 with CBP. Administration of aminoguanidine concomitant with increasing glucose levels in our in vitro model system protected from glucose-induced “silencing” of the podocalyxin gene, suggesting that AGEs play an important role in suppressing its expression in diabetic conditions.
Role of pendrin in iodide balance: going with the flow
Authors: Kim Young Hee , Pham Truyen D. , Zheng Wencui , Hong Seongun , Baylis Christine , Pech Vladimir , Beierwaltes William H. , Farley Donna B. , Braverman Lewis E. , Verlander Jill W. , Wall Susan M.
Published: July 2009

Pendrin is expressed in the apical regions of type B and non-A, non-B intercalated cells, where it mediates Cl−absorption and HCO3−secretion through apical Cl−/HCO3−exchange. Since pendrin is a robust I−transporter, we asked whether pendrin is upregulated with dietary I−restriction and whether it modulates I−balance. Thus I−balance was determined in pendrin null and in wild-type mice. Pendrin abundance was evaluated with immunoblots, immunohistochemistry, and immunogold cytochemistry with morphometric analysis. While pendrin abundance was unchanged when dietary I−intake was varied over the physiological range, I−balance differed in pendrin null and in wild-type mice. Serum I−was lower, while I−excretion was higher in pendrin null relative to wild-type mice, consistent with a role of pendrin in renal I−absorption. Increased H2O intake enhanced differences between wild-type and pendrin null mice in I−balance, suggesting that H2O intake modulates pendrin abundance. Raising water intake from ∼4 to ∼11 ml/day increased the ratio of B cell apical plasma membrane to cytoplasm pendrin label by 75%, although circulating renin, aldosterone, and serum osmolality were unchanged. Further studies asked whether H2O intake modulates pendrin through the action of AVP. We observed that H2O intake modulated pendrin abundance even when circulating vasopressin levels were clamped. We conclude that H2O intake modulates pendrin abundance, although not likely through a direct, type 2 vasopressin receptor-dependent mechanism. As water intake rises, pendrin becomes increasingly critical in the maintenance of Cl−and I−balance.
Allopurinol, rutin, and quercetin attenuate hyperuricemia and renal dysfunction in rats induced by fructose intake: renal organic ion transporter involvement
Authors: Hu Qing-Hua , Wang Chuang , Li Jian-Mei , Zhang Dong-Mei , Kong Ling-Dong
Published: July 2009

Fructose consumption has been recently related to an epidemic of metabolic syndrome, and hyperuricemia plays a pathogenic role in fructose-induced metabolic syndrome. Fructose-fed rats showed hyperuricemia and renal dysfunction with reductions of the urinary uric acid/creatinine ratio and fractional excretion of uric acid (FEur), as well as other features of metabolic syndrome. Lowering serum uric acid levels with allopurinol, rutin, and quercetin increased the urinary uric acid/creatinine ratio and FEurand attenuated other fructose-induced metabolic abnormalities in rats, demonstrating that hyperuricemia contributed to the deficiency of renal uric acid excretion in this model. Furthermore, we found that fructose upregulated the expression levels of rSLC2A9v2 and renal-specific transporter (rRST), downregulated the expression levels of organic anion transporters (rOAT1 and rUAT) and organic cation transporters (rOCT1 and rOCT2), with the regulators prostaglandin E2(PGE2) elevation and nitric oxide (NO) reduction in rat kidney. Allopurinol, rutin, and quercetin reversed dysregulations of these transporters with PGE2reduction and NO elevation in the kidney of fructose-fed rats. These results suggested that dysregulations of renal rSLC2A9v2, rRST, rOAT1, rUAT, rOCT1, and rOCT2 contributed to fructose-induced hyperuricemia and renal dysfunction. Therefore, these renal transporters may represent novel therapeutic targets for the treatment of hyperuricemia and renal dysfunction in fructose-induced metabolic syndrome.
Neuromechanical matching of drive in the scalene muscle of the anesthetized rabbit
Authors: Legrand Alexandre , Majcher Melanie , Joly Emma , Bonaert Adeline , Gevenois Pierre Alain
Published: July 2009

The scalene is a primary respiratory muscle in humans; however, in dogs, EMG activity recorded from this muscle during inspiration was reported to derive from underlying muscles. In the present studies, origin of the activity in the medial scalene was tested in rabbits, and its distribution was compared with the muscle mechanical advantage. We assessed in anesthetized rabbits the presence of EMG activity in the scalene, sternomastoid, and parasternal intercostal muscles during quiet breathing and under resistive loading, before and after denervation of the scalene and after its additional insulation. At rest, activity was always recorded in the parasternal muscle and in the scalene bundle inserting on the third rib (medial scalene). The majority of this activity disappeared after denervation. In the bundle inserting on the fifth rib (lateral scalene), the activity was inconsistent, and a high percentage of this activity persisted after denervation but disappeared after insulation from underlying muscle layers. The sternomastoid was always silent. The fractional change in muscle length during passive inflation was then measured. The mean shortening obtained for medial and lateral scalene and parasternal intercostal was 8.0 ± 0.7%, 5.5 ± 0.5%, and 9.6 ± 0.1%, respectively, of the length at functional residual capacity. Sternomastoid muscle length did not change significantly with lung inflation. We conclude that, similar to that shown in humans, respiratory activity arises from scalene muscles in rabbits. This activity is however not uniformly distributed, and a neuromechanical matching of drive is observed, so that the most effective part is also the most active.
In vivo intramuscular fascicle-aponeuroses dynamics of the human medial gastrocnemius during plantarflexion and dorsiflexion of the foot
Authors: Shin David D. , Hodgson John A. , Edgerton V. Reggie , Sinha Shantanu
Published: July 2009

Velocity-encoded phase-contrast magnetic resonance (MR) imaging techniques and a computer-controlled MR-compatible foot pedal device were used to investigate the medial gastrocnemius muscle and aponeurosis deformations during passive and active eccentric movements of the plantarflexors. Intrafascicular strain, measured as the ratio of strain in the fascicle segment at its insertion to strain at its origin, was nonuniform along the proximodistal axis of the muscle ( P < 0.01), progressively increasing from the proximal to distal direction. The high intrafascicular strain regions appeared to correlate with the muscle regions that are likely to encounter high stress concentrations, i.e., the regions where the muscle physiological cross section decreases close to the tendons. The architectural gear ratio, i.e., the mechanical amplification ratio of fascicle length displacement to that of tendon/aponeuroses in a pennate muscle, also exhibited significant regional differences, with the highest ratios in the proximal region of the muscle accompanied by a higher initial pennation angle and a larger range of fascicular rotation about the origin. Values close to unity in the distal region of the muscle suggest that the aponeurosis separation may decrease in this region. Fascicle length and pennation angle changes were significantly influenced by force generation in the muscle, probably due to a shortening of the loaded muscle fibers relative to a passive condition. Overall, our data illustrate significant proximodistal intramuscular heterogeneity as supported by a regionally variable end-to-end strain ratio of fascicles and angle changes in the medial gastrocnemius muscle during passive and active ankle movements. These observations emphasize the need to reassess current conceptual models of muscle-tendon mechanics.
Statistics of Natural Movements Are Reflected in Motor Errors
Authors: Howard Ian S. , Ingram James N. , Körding Konrad P. , Wolpert Daniel M.
Published: July 2009

Humans use their arms to engage in a wide variety of motor tasks during everyday life. However, little is known about the statistics of these natural arm movements. Studies of the sensory system have shown that the statistics of sensory inputs are key to determining sensory processing. We hypothesized that the statistics of natural everyday movements may, in a similar way, influence motor performance as measured in laboratory-based tasks. We developed a portable motion-tracking system that could be worn by subjects as they went about their daily routine outside of a laboratory setting. We found that the well-documented symmetry bias is reflected in the relative incidence of movements made during everyday tasks. Specifically, symmetric and antisymmetric movements are predominant at low frequencies, whereas only symmetric movements are predominant at high frequencies. Moreover, the statistics of natural movements, that is, their relative incidence, correlated with subjects' performance on a laboratory-based phase-tracking task. These results provide a link between natural movement statistics and motor performance and confirm that the symmetry bias documented in laboratory studies is a natural feature of human movement.
Flexible Learning of Natural Statistics in the Human Brain
Authors: Schwarzkopf D. Samuel , Zhang Jiaxiang , Kourtzi Zoe
Published: July 2009

The ability to detect and identify targets in cluttered scenes is a critical skill for survival and interactions. To solve this challenge the brain has optimized mechanisms for capitalizing on frequently occurring regularities in the environment. Although evolution and development have been suggested to shape the brain's architecture in a manner that resembles these natural statistics, we provide novel evidence that short-term experience in adulthood may modify the brain's functional organization to support integration of signals atypical of shape contours in natural scenes. Although collinearity is a prevalent principle for perceptual integration in natural scenes, we show that observers learn to exploit other image regularities (i.e., orthogonal alignments of segments at an angle to the contour path) that typically signify discontinuities. Combining behavioral and functional MRI measurements, we demonstrate that this flexible learning is mediated by changes in the neural representations of behaviorally relevant image regularities primarily in dorsal visual areas. These changes in neural sensitivity are in line with changes in perceptual sensitivity for the detection of orthogonal contours and are evident only in observers that show significant performance improvement. In contrast, changes in the activation extent in frontoparietal regions are evident independent of performance changes, may support the detection of salient regions, and modulate perceptual integration in occipitotemporal areas in a top-down manner. Thus experience at shorter timescales in adulthood supports the adaptive functional optimization of visual circuits for flexible interpretation of natural scenes.
Changes in Performance Monitoring During Sensorimotor Adaptation
Authors: Anguera Joaquin A. , Seidler Rachael D. , Gehring William J.
Published: July 2009

Error detection and correction are essential components of motor skill learning. These processes have been well characterized in cognitive psychology using electroencephalography (EEG) to record an event-related potential (ERP) called error-related negativity (ERN). However, it is unclear whether this ERP component is sensitive to the magnitude of the error made in a sensorimotor adaptation task. In the present study, we tested the function of error-related activity in a visuomotor adaptation task. To examine whether error size is reflected in the ERP, two groups of participants adapted manual aiming movements to either a small (30°) or large (45°) rotation of the visual feedback display. Each participant's trials were sorted into large and small error trials using a median split to examine potential error magnitude waveform differences. We also examined these trial types at the early and late stages of adaptation. There were no group differences for the behavioral or neural measures; however, waveforms from large error trials were significantly different from small error trials. The waveforms also changed as a function of practice as early adaptation waveforms were larger than late adaptation waveforms. The observed ERP component reflected differences in error magnitude with the amount of activity corresponding to the size of the error. Movement monitoring potentials likely affected the frequency and time course of the waveform so that it did not resemble the typical ERN; however, error-related activity was still distinguishable. The present findings are discussed in terms of current theories of the ERN as well as skill acquisition.
Premotor Interneurons in the Local Control of Stepping Motor Output for the Stick Insect Single Middle Leg
Authors: von Uckermann Géraldine , Büschges Ansgar
Published: July 2009

In insect walking systems, nonspiking interneurons (NSIs) play an important role in the control of posture and movement. As such NSIs are known to contribute to state-dependent modifications in processing of proprioceptive signals from the legs. For example, NSIs process a flexion of the femur-tibia (FTi) joint signaled by the femoral chordotonal organ (fCO) such that the stance phase motor output is reinforced in the active locomotor system. This phenomenon representing a reflex reversal is the first part of the “active reaction” (AR) and was hypothesized to functionally represent a major control feature by which sensory feedback supports stance generation. As NSIs are known to contribute to the AR, the question arises, whether they serve similar functions during stepping and whether the AR is generally part of the control system for walking. We studied these issues in vivo, in a single leg preparation of Carausius morosus with the leg kinematics being confined to changes in one plane, along the coxa-trochanteral and the FTi-joint. Following kinematic analysis, identified NSIs (E1-E8, I1, I2, and I4) were recorded intracellularly during single leg stepping at different velocities. We detected clear similarities between the activity pattern of NSIs during single leg stepping and their responses to fCO-stimulation during the generation of the AR. This strongly supports the notion that the motor output generated during the AR reflects part of the control regime for stepping. Furthermore, our experiments revealed that alterations in stepping velocity result from modifications in the activity of the premotor NSIs involved in stance phase generation.
Inhibitory Effect of Somatostatin-14 on L-Type Voltage-Gated Calcium Channels in Cultured Cone Photoreceptors Requires Intracellular Calcium
Authors: Jian Kuihuan , Barhoumi Rola , Ko Michael L. , Ko Gladys Y.-P.
Published: July 2009

The inhibitory effects of somatostatin have been well documented for many physiological processes. The action of somatostatin is through G-protein-coupled receptor-mediated second-messenger signaling, which in turn affects other downstream targets including ion channels. In the retina, somatostatin is released from a specific class of amacrine cells. Here we report that there was a circadian phase-dependent effect of somatostatin-14 (SS14) on the L-type voltage-gated calcium channels (L-VGCCs) in cultured chicken cone photoreceptors, and our study reveals that this process is dependent on intracellular calcium stores. Application of 500 nM SS14 for 2 h caused a decrease in L-VGCC currents only during the subjective night but not the subjective day. We then explored the cellular mechanisms underlying the circadian phase-dependent effect of SS14. The inhibitory effect of SS14 on L-VGCCs was mediated through the pertussis-toxin-sensitive G-protein-dependent somatostatin receptor 2 (sst2). Activation of sst2 by SS14 further activated downstream signaling involving phospholipase C and intracellular calcium stores. Mobilization of intracellular Ca2+was required for somatostatin induced inhibition of photoreceptor L-VGCCs, suggesting that somatostatin plays an important role in the modulation of photoreceptor physiology.
Contribution of Hindpaw Cutaneous Inputs to the Control of Lateral Stability During Walking in the Cat
Authors: Bolton D.A.E. , Misiaszek J. E.
Published: July 2009

To delineate the role of cutaneous feedback from the paws in the regulation of balance during walking, we compared the corrective responses of cats to lateral support surface translation before and after cutaneous denervation of the hindpaws. In addition, we compared characteristics of undisturbed walking before and after denervation. Electromyographic and kinematic data were collected from three cats trained to walk across a walkway, the central portion of which could be unexpectedly translated laterally in either direction. Following denervation, all of the cats changed their step width, lowered their pelvis, and spent more time with the hindlegs in double-support when walking across the walkway. When displaced by lateral support surface translations, the denervated cats made larger lateral steps and required more than a single step to regain balance. However, none of the cats fell following the denervation. The appearance and latency of the responses evoked in the hindleg muscles by the perturbations were unaffected by the denervation. However, the amplitude of these responses was affected by the loss of cutaneous inputs. Responses evoked at paw contact were significantly reduced in most muscles in the absence of cutaneous input, whereas responses evoked at end of stance revealed significant increases in gluteus medius activity with little influence on the activity of other muscles. Therefore the loss of cutaneous inputs leads to instability during gait. Although cutaneous feedback from the hindpaws is not essential for triggering corrective responses to support surface disturbances, it appears that cutaneous inputs are important for scaling the responses initiated by other cues.
Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101
Authors: Vidyasagar Malashetty , Prakash S. , Litchfield Carol , Sreeramulu K.
Published: July 2009

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116‐fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 °C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 °C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.
The archaeal origins of the eukaryotic translational system
Authors: Hartman Hyman , Favaretto Paola , Smith Temple F.
Published: July 2009

Among the 78 eukaryotic ribosomal proteins, eleven are specific to Eukarya, 33 are common only to Archaea and Eukarya and 34 are homologous (at least in part) to those of both Bacteria and Archaea. Several other translational proteins are common only to Eukarya and Archaea (e.g., IF2a, SRP19, etc.), whereas others are shared by the three phyla (e.g., EFTu/EF1A and SRP54).

Although this and other analyses strongly support an archaeal origin for a substantial fraction of the eukaryotic translational machinery, especially the ribosomal proteins, there have been numerous unique and ubiquitous additions to the eukaryotic translational system besides the 11 unique eukaryotic ribosomal proteins. These include peptide additions to most of the 67 archaeal homolog proteins, rRNA insertions, the 5.8S RNA and the Alu extension to the SRP RNA. Our comparative analysis of these and other eukaryotic features among the three different cellular phylodomains supports the idea that an archaeal translational system was most likely incorporated by means of endosymbiosis into a host cell that was neither bacterial nor archaeal in any modern sense. Phylogenetic analyses provide support for the timing of this acquisition coinciding with an ancient bottleneck in prokaryotic diversity.
An analysis of amino acid sequences surrounding archaeal glycoprotein sequons
Authors: Abu-Qarn Mehtap , Eichler Jerry
Published: July 2009

Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N‐glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N‐glycosylation takes place at the Asn residues of Asn‐X‐Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N‐glycosylation made by sequon‐bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N‐glycosylation.