bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanical stress induces tumor necrosis factor-α production through Ca2+ release-dependent TLR2 signaling
Authors: Kim Han Geun , Kim Joo Yun , Gim Min Geun , Lee Jung Min , Chung Dae Kyun
Published: June 2008

We studied centrifugation-mediated mechanical stress-induced tumor necrosis factor-α (TNF-α) production in the monocyte-like cell line THP-1. The induction of TNF-α by mechanical stress was dependent on the centrifugation speed and produced the highest level of TNF-α after 1 h of stimulation. TNF-α production returned to normal levels after 24 h of stimulation. Mechanical stress also induced Toll-like receptor-2 (TLR2) mRNA in proportion to the expression of TNF-α. The inhibition of TLR2 signaling by dominant negative myeloid differentiation factor 88 (MyD88) blocked TNF-α expression response to mechanical stress. After transient overexpression of TLR2 in HEK-293 cells, mechanical stress induced TNF-α mRNA production. Interestingly, mechanical stress activated the c-Src-dependent TLR2 phosphorylation, which is necessary to induce Ca2+ fluxes. When THP-1 cells were pretreated with BAPTA-AM, thapsigargin, and NiCl2·6H2O, followed by mechanical stimulation, both TLR2 and TNF-α production were inhibited, indicating that centrifugation-mediated mechanical stress induces both TLR2 and TNF-α production through Ca2+ releases from intracellular Ca2+ stores following TLR2 phosphorylation. In addition, TNF-α treatment in THP-1 cells induced TLR2 production in response to mechanical stress, whereas the preincubation of anti-TNF-α antibody scarcely induced the mechanical stress-mediated production of TLR2, indicating that TNF-α produced by mechanically stimulated THP-1 cells affected TLR2 production. We concluded that TNF-α production induced by centrifugation-mediated mechanical stress is dependent on MyD88-dependent TLR2 signaling that is associated with Ca2+ release and that TNF-α production induced by mechanical stress affects TLR2 production.
Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1
Authors: Tseng I-Chu , Chou Feng-Pai , Su Sheng-Feng , Oberst Michael , Madayiputhiya Nandakumar , Lee Ming-Shyue , Wang Jehng-Kang , Sloane David E. , Johnson Michael , Lin Chen-Yong
Published: June 2008

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110 kDa in human milk, which contained no HAI-1 and resisted dissociation in boiling SDS in the absence of reducing agents. These complexes were further purified and dissociated into 80-kDa and 45-kDa fragments by treatment with reducing agents. Proteomic and immunological methods identified the 45-kDa fragment as the noncatalytic domains of matriptase and the 80-kDa fragment as the matriptase serine protease domain covalently linked to one of three different secreted serpin inhibitors: antithrombin III, α1-antitrypsin, and α2-antiplasmin. Identification of matriptase-serpin inhibitor complexes provides evidence for the first time that the proteolytic activity of matriptase, from those cells that express no or low levels of HAI-1, may be controlled by secreted serpins.
Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells
Authors: Chernysh Olga , Condrescu Madalina , Reeves John P.
Published: June 2008

High concentrations of cytosolic Na+ ions induce the time-dependent formation of an inactive state of the Na+/Ca2+ exchanger (NCX), a process known as Na+-dependent inactivation. NCX activity was measured as Ca2+ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na+-dependent inactivation. As expected, 1) Na+-dependent inactivation was promoted by high cytosolic Na+ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na+-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na+-dependent inactivation in the WT exchanger, 3) Na+-dependent inactivation did not increase the half-maximal cytosolic Ca2+ concentration for allosteric Ca2+ activation, 4) Na+-dependent inactivation was not reversed by high cytosolic Ca2+ concentrations, and 5) Na+-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca2+ concentration. Thus Na+-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na+-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca2+ influx during ischemia.
The effect of rapamycin on DNA synthesis in multiple tissues from late gestation fetal and postnatal rats
Authors: Sanders Jennifer A. , Lakhani Alisha , Phornphutkul Chanika , Wu Ke-Ying , Gruppuso Philip A.
Published: June 2008

Rapamycin is a potent antiproliferative agent that arrests cells in the G1 phase of the cell cycle through a variety of mechanisms involving the inhibition of the mammalian target of rapamycin (mTOR) pathway. The majority of normal cells in culture are sensitive to the cytostatic effects of rapamycin, whereas the growth of many malignant cells and tumors is rapamycin resistant. We had shown previously that hepatic DNA synthesis in the late gestation rat fetus is rapamycin resistant even though signaling through the mTOR/S6 kinase (S6K) pathway is attenuated. On the basis of this finding, we went on to characterize the response to rapamycin in a spectrum of tissues during late gestation and the early postnatal period in the rat. We found that rapamycin had no effect on DNA synthesis in major organs such as heart, intestine, and kidney in the fetal and early postnatal rat despite a marked attenuation in the phosphorylation of ribosomal protein S6. In contrast, the proliferation of mature hepatocytes during liver regeneration was highly sensitive to rapamycin. These data indicate that basal cellular proliferation in a wide variety of tissues is rapamycin resistant and occurs independently of mTOR/S6K signaling. Furthermore, the well-characterized effects of rapamycin in tissue culture systems are not recapitulated in the asynchronous cell proliferation that accompanies normal growth and tissue remodeling.
Effect of pioglitazone treatment on endoplasmic reticulum stress response in human adipose and in palmitate-induced stress in human liver and adipose cell lines
Authors: Das Swapan K. , Chu Winston S. , Mondal Ashis K. , Sharma Neeraj K. , Kern Philip A. , Rasouli Neda , Elbein Steven C.
Published: June 2008

Obesity and elevated cytokine secretion result in a chronic inflammatory state and may cause the insulin resistance observed in type 2 diabetes. Recent studies suggest a key role for endoplasmic reticulum stress in hepatocytes and adipocytes from obese mice, resulting in reduced insulin sensitivity. To address the hypothesis that thiazolidinediones, which improve peripheral insulin sensitivity, act in part by reducing the endoplasmic reticulum stress response, we tested subcutaneous adipose tissue from 20 obese volunteers treated with pioglitazone for 10 wk. We also experimentally induced endoplasmic reticulum stress using palmitate, tunicamycin, and thapsigargin in the human HepG2 liver cell line with or without pioglitazone pretreatment. We quantified endoplasmic reticulum stress response by measuring both gene expression and phosphorylation. Pioglitazone significantly improved insulin sensitivity in human volunteers ( P = 0.002) but did not alter markers of endoplasmic reticulum stress. Differences in pre- and posttreatment endoplasmic reticulum stress levels were not correlated with changes in insulin sensitivity or body mass index. In vitro, palmitate, thapsigargin, and tunicamycin but not oleate induced endoplasmic reticulum stress in HepG2 cells, including increased transcripts CHOP, ERN1, GADD34, and PERK, and increased XBP1 splicing along with phosphorylation of eukaryotic initiation factor eIF2α, JNK1, and c- jun. Although patterns of endoplasmic reticulum stress response differed among palmitate, tunicamycin, and thapsigargin, pioglitazone pretreatment had no significant effect on any measure of endoplasmic reticulum stress, regardless of the inducer. Together, our data suggest that improved insulin sensitivity with pioglitazone is not mediated by a reduction in endoplasmic reticulum stress.
Altered mesenteric venous capacitance and volume pooling in cirrhotic rats are mediated by nitric oxide
Authors: Li Yang , Liu Hongqun , Gaskari Seyed Ali , Tyberg John V. , Lee Samuel S.
Published: June 2008

In cirrhosis, despite augmented blood volume, effective circulating volume is decreased. This implies abnormal regulation of blood volume, i.e., venous pooling. Because gut veins are the main blood reservoir, we studied mesenteric venous capacitance and compliance in a rat model of cirrhosis. Cirrhosis was induced by bile duct ligation (4 wk). Controls were sham operated. Changes in first-order mesenteric vein diameters induced by drugs, hemorrhage, and stepwise increases in portal pressure (inflatable cuff) were directly observed by intravital microscopy. Effects of nitric oxide on responses to acute graded hemorrhage were studied by use of selective NO synthase (NOS) isoform inhibitors. Pressures were related to diameters to assess capacitance and compliance. Compared with controls, cirrhotic rats demonstrated increased mesenteric venous capacitance and decreased compliance. Norepinephrine induced venoconstriction but did not affect compliance. Prazosin markedly diminished compliance in controls but not cirrhotics. Conversely, the nonspecific NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) decreased compliance in cirrhotics, but not controls. Tetrodotoxin venodilated controls, venoconstricted cirrhotics, and markedly decreased compliance in both groups. When hemorrhaged, controls rapidly venoconstricted to compensate for initial hypotension, whereas cirrhotic rats remained hypotensive because venoconstriction was severely blunted. Pretreatment with l-NAME or the selective neuronal NOS inhibitors S-methyl-l-thiocitrulline and 7-nitroindazole normalized the homeostatic responses of cirrhotic rats, whereas the selective endothelial-constitutive NOS inhibitor N-iminoethyl-l-ornithine did not affect the response. In conclusion, mesenteric veins of cirrhotic rats showed enhanced capacitance, attenuated compensatory constrictive response to hemorrhage, and decreased compliance. The first two abnormalities were caused by neuronal NOS-derived nitric oxide.
Interaction of CD44 and hyaluronic acid enhances biliary epithelial proliferation in cholestatic livers
Authors: He Yao , Wu Gordon D. , Sadahiro Tomohito , Noh Sang-Ik , Wang Hong , Talavera Dodanim , Wang Haimei , Vierling John M. , Klein Andrew S.
Published: June 2008

Biliary epithelia express high levels of CD44 in hepatobiliary diseases. The role of CD44-hyaluronic acid interaction in biliary pathology, however, is unclear. A rat model of hepatic cholestasis induced by bile duct ligation was employed for characterization of hepatic CD44 expression and extracellular hyaluronan distribution. Cell culture experiments were employed to determine whether hyaluronan can regulate cholangiocyte growth through interacting with adhesion molecule CD44. Biliary epithelial cells were found to express the highest level of CD44 mRNA among four major types of nonparenchymal liver cells, including Kupffer, hepatic stellate, and liver sinusoidal endothelial cells isolated from cholestatic livers. CD44-positive biliary epithelia lining the intrahepatic bile ducts were geographically associated with extracellular hyaluronan accumulated in the portal tracts of the livers, suggesting a role for CD44 and hyaluronan in the development of biliary proliferation. Cellular proliferation assays demonstrated that cholangiocyte propagation was accelerated by hyaluronan treatment and antagonized by small interfering RNA CD44 or anti-CD44 antibody. The study provides compelling evidence to suggest that proliferative biliary epithelia lining the intrahepatic bile ducts are a prime source of hepatic CD44. CD44-hyaluronan interaction, by enhancing biliary proliferation, may play a pathogenic role in the development of cholestatic liver diseases.
Acid, Bile, and CDX: the ABCs of making Barrett's metaplasia
Authors: Souza Rhonda F. , Krishnan Kumar , Spechler Stuart Jon
Published: June 2008

Barrett's esophagus, a squamous-to-columnar cell metaplasia that develops as a result of chronic gastroesophageal reflux disease (GERD), is a risk factor for esophageal adenocarcinoma. The molecular events underlying the pathogenesis of Barrett's metaplasia are poorly understood, but recent studies suggest that interactions among developmental signaling pathways, morphogenetic factors, and Caudal homeobox (Cdx) genes play key roles. Strong expression of Cdx genes normally is found in the intestine but not in the esophagus and stomach. When mice are genetically engineered so that their gastric cells express Cdx, the stomach develops a metaplastic, intestinal-type epithelium similar to that of Barrett's esophagus. Exposure to acid and bile has been shown to activate the Cdx promoter in certain esophageal cell lines, and Cdx expression has been found in inflamed esophageal squamous epithelium and in the specialized intestinal metaplasia of Barrett's esophagus. Barrett's metaplasia must be sustained by stem cells, which might be identified by putative, intestinal stem cell markers like leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and doublecortin and CaM kinase-like-1 (DCAMKL-1). Emerging concepts in tumor biology suggest that Barrett's cancers may develop from growth-promoting mutations in metaplastic stem cells or their progenitor cell progeny. This report reviews the roles of developmental signaling pathways and the Cdx genes in the development of normal gut epithelia and the potential mechanisms whereby GERD may induce the esophageal expression of Cdx genes and other morphogenetic factors that mediate the development of Barrett's metaplasia. The role of stem cells in the development of metaplasia and in carcinogenesis and the potential for therapies directed at those stem cells also is addressed.
Short-term secondhand smoke exposure decreases heart rate variability and increases arrhythmia susceptibility in mice
Authors: Chen Chao-Yin , Chow Drin , Chiamvimonvat Nipavan , Glatter Kathryn A. , Li Ning , He Yuxia , Pinkerton Kent E. , Bonham Ann C.
Published: June 2008

Exposure to secondhand smoke (SHS), a major indoor air pollutant, is linked to increased cardiovascular morbidity and mortality, including cardiac arrhythmias. However, the mechanisms underlying the epidemiological findings are not well understood. Impaired cardiac autonomic function, indexed by reduced heart rate variability (HRV), may represent an underlying cause. The present study takes advantage of well-defined short-term SHS exposure (3 days, 6 h/day) on HRV and the susceptibility to arrhythmia in mice. With the use of electrocardiograph telemetry recordings in conscious mice, HRV parameters in the time domain were measured during the night after each day of exposure and 24 h after 3 days of exposure to either SHS or filtered air. The susceptibility to arrhythmia was determined after 3 days of exposure. Exposure to a low concentration of SHS [total suspended particle (TSP), 2.4 ± 3.2; and nicotine, 0.3 ± 0.1 mg/m3] had no significant effect on HRV parameters. In contrast, the exposure to a higher but still environmentally relevant concentration of SHS (TSP, 30 ± 1; and nicotine, 5 ± 1 mg/m3) significantly reduced HRV starting after the first day of exposure and continuing 24 h after the last day of exposure. Moreover, the exposed mice showed a significant increase in ventricular arrhythmia susceptibility and atrioventricular block. The data suggest that SHS exposure decreased HRV beyond the exposure period and was associated with an increase in arrhythmia susceptibility. The data provide insights into possible mechanisms underlying documented increases in cardiovascular morbidity and mortality in humans exposed to SHS.
Antagonism of corticotrophin-releasing factor receptors in the fourth ventricle modifies responses to mild but not restraint stress
Authors: Miragaya Joanna R. , Harris Ruth B. S.
Published: June 2008

Repeated restraint stress (RRS; 3 h of restraint on 3 consecutive days) in rodents produces temporary hypophagia, but a long-term downregulation of body weight. The mild stress (MS) of an intraperitoneal injection of saline and housing in a novel room for 2 h also inhibits food intake and weight gain, but the effects are smaller than for RRS. Previous exposure to RRS exaggerates hypophagia, glucocorticoid release, and anxiety-type behavior caused by MS. Here we tested the involvement of brain stem corticotrophin-releasing factor receptors (CRFR) in mediating energetic and glucocorticoid responses to RRS or MS and in promoting stress hyperresponsiveness in RRS rats. Administration of 1.3 nmol αhCRF(9-41), a nonspecific CRFR antagonist, exaggerated hypophagia and weight loss in both RRS and MS rats, whereas 0.26 nmol had no effect in RRS or MS rats. In contrast, 2 nmol of the nonspecific antagonist astressin had no effect on weight loss or hypersensitivity to subsequent MS in RRS rats, but blocked weight loss and inhibition of food intake caused by MS alone. MS rats infused with 3 nmol antisauvagine-30, a CRFR2 antagonist, did not lose weight in the 48 h after MS, but 0.3 nmol did not prevent weight loss in MS rats. These data suggest that inhibition of food intake and weight loss induced by RRS or by MS involve different pathways, with hindbrain CRFR mediating the effect of MS on body weight and food intake. Hindbrain CRFR do not appear to influence stress-induced corticosterone release in RRS rats.
Effects of age on thermoregulatory responses during cold exposure in a nonhuman primate, Microcebus murinus
Authors: Terrien J. , Zizzari P. , Bluet-Pajot M.-T. , Henry P.-Y. , Perret M. , Epelbaum J. , Aujard F.
Published: June 2008

Cold resistance appears altered with aging. Among existing hypotheses, the impaired capacity in response to cold could be related to an altered regulation of plasma IGF-1 concentration. The combined effects of age and cold exposure were studied in a short-living primate, the gray mouse lemur ( Microcebus murinus), which adjusts its energy balance using a daily torpor phase, to avoid high energy cost of normothermia maintenance. Changes in body mass, core temperature, locomotor activity, and caloric intake were monitored under 9-day exposures to 25°C and 12°C in captive animals in winter conditions. Short-term (after 2 days) and long-term (after 9 days) cold-induced changes in IGF-1 levels were also evaluated. In thermoneutral conditions (25°C), general characteristics of the daily rhythm of core temperature were preserved with age. At 12°C, age-related changes were mainly characterized by a deeper hypothermia and an increased frequency of torpor phases, associated with a loss of body mass. A short-term cold-induced decrease in plasma IGF-1 levels was observed. IGF-1 levels returned to basal values after 9 days of cold exposure. No significant effect of age could be evidenced on IGF-1 response. However, IGF-1 levels of cold-exposed aged animals were negatively correlated with the frequency of daily torpor. Responses exhibited by aged mouse lemurs exposed to cold revealed difficulties in the maintenance of normothermia and energy balance and might involve modulations of IGF-1 levels.
Long-lasting breaches in the bladder epithelium lead to storage dysfunction with increase in bladder PGE2 levels in the rat
Authors: Shioyama Rikiya , Aoki Yoshitaka , Ito Hideaki , Matsuta Yosuke , Nagase Keiko , Oyama Nobuyuki , Miwa Yoshiji , Akino Hironobu , Imamura Yoshiaki , Yokoyama Osamu
Published: June 2008

Increase in bladder mucosal permeability can be reproduced by intravesical administration of protamine sulfate (PS); however, the influence of PS once administered into the bladder disappears within several days. We developed a chronic animal model of urothelial injury using PS. Insertion of a polyethylene catheter through the bladder dome was performed in female Wistar rats. The other end of the catheter was connected to an osmotic pump for continuous delivery of PS or vehicle for 2 wk. Urinary frequency (UF) and voided volume (VV) were measured in the metabolic cage. The fifth group of rats received a high dose of PS (10 mg/ml) for 2 wk and were followed for a further 2 wk without PS. The sixth group received a high dose of PS for 2 wk and loxoprofen (0.1 mg·kg−1·day−1) for 4 wk. UF was increased, and VV was reduced in rats treated with a high dose of PS but not changed in rats treated with a vehicle or a low dose of PS (1 mg/ml). UF was further increased in the fifth group, while unchanged in the sixth group. Histological sections in rats treated with a high dose of PS demonstrated a loss of the upper layer of urothelial cells and an increased number of mast cells. PGE2 level in the bladder was significantly elevated in the fifth group. These results indicate that chronic urotherial injury leads to an increase in UF and a decrease in VV. Increased PGE2 level in the bladder is likely to be associated with long-lasting storage dysfunction.
Sex-related changes in cardiac function following myocardial infarction in mice
Authors: Shioura Krystyna M. , Geenen David L. , Goldspink Paul H.
Published: June 2008

Recent awareness of cardiovascular diseases as a number one killer of the middle-aged women has prompted interest in sex differences leading to heart failure (HF). Therefore, we evaluated cardiac function in female and male mice following myocardial infarction (MI) using the Millar pressure-volume (P-V) conductance system in vivo, at time points corresponding to early (2 wk), late compensatory hypertrophy (4 wk), and decompensation (10 wk) to HF. A significant deterioration of the load dependent and independent hemodynamic measurements occurred in both female and male mice during the early phase of hypertrophy. Later, compensatory hypertrophy was marked by a normalization of volumes to control levels in females compared with males. The most notable differences between sexes occurred in the measurements of cardiac contractility during the decompensation to HF. In females, there was a significant improvement in contractility compared with males, which was apparent in the load-independent measurements of preload recruitable stroke work (10 wk post-MI, female = 48.7 ± 8.0 vs. male = 25.2 ± 1.8 mmHg, P < 0.05) and maximum dP/d t vs. maximum end-diastolic volume (10 wk post-MI, female=359 ± 58 vs. male=149 ± 28 mmHg·s−1·μl−1, P < 0.05). Despite these differences, there were no differences in the heart weight to body weight ratio and infarct size between the sexes. These data demonstrate that compensatory hypertrophy is associated with an improvement in contractility and a delayed decompensation to HF in females. However, compensatory hypertrophy in males appears to be undermined by a steady decline in contractility associated with decompensation to HF.
Parathyroid hormone-related protein interacts with vascular endothelial growth factor to promote fibrogenesis in the obstructed mouse kidney
Authors: Ardura Juan A. , Berruguete Raúl , Rámila David , Alvarez-Arroyo M. Victoria , Esbrit Pedro
Published: June 2008

Parathyroid hormone-related protein (PTHrP) interacts with vascular endothelial growth factor (VEGF) in osteoblasts. Since both PTHrP and VEGF have both proinflammatory and profibrogenic features, we assessed here whether these factors might act in concert to promote fibrogenesis in the obstructed kidney. VEGF receptor (VEGFR)-1 was upregulated, while VEGFR-2 was downregulated (at both mRNA and protein levels) in the mouse kidney within 2–6 days after ureteral obstruction. VEGF protein levels also increased in the obstructed kidney at the latter time. Moreover, this VEGF and VEGFR-1 upregulation was higher in mice overexpressing PTHrP in the proximal tubule than in control littermates. These changes were associated with higher fibronectin mRNA expression and α-smooth muscle actin (α-SMA) and integrin-linked kinase (ILK) immunostaining and lower apoptotic tubulointerstitial cells in the mouse obstructed kidney than in control littermates. Pretreatment with a neutralizing anti-VEGF antibody reversed these responses in the obstructed kidney of both types of mice. In vitro, PTHrP-(1-36) increased (maximal 2-fold vs. basal, at 100 nM) α-SMA and ILK protein expression and decreased E-cadherin protein levels in renal tubuloepithelial mouse cortical tubule and normal rat kidney (NRK) 52E cells. PTHrP-(1-36) also decreased cyclosporine A- and/or osmotic stress-induced apoptosis in these cells and in renal fibroblastic NRK 49F cells. These effects elicited by PTHrP-(1-36) were associated with both VEGF and VEGFR-1 upregulation, and abolished by the anti-VEGF antibody. Collectively, these findings strongly suggest that VEGF acts as an important mediator of PTHrP to promote fibrogenesis in the obstructed kidney.
PTH-mediated regulation of Na+-K+-ATPase requires Src kinase-dependent ERK phosphorylation
Authors: Khundmiri Syed J. , Ameen Mohammed , Delamere Nicholas A. , Lederer Eleanor D.
Published: June 2008

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the α1-subunit through ERK-dependent phosphorylation and translocation of protein kinase Cα (PKCα). On the basis of previous studies, we postulated that PTH regulates sodium pump activity through Src kinase, PLC, and calcium-dependent ERK phosphorylation. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKCα were prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacological inhibition of PLA2 did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKCα translocation. Silencing the expression of cytosolic or calcium-independent PLA2 also prevented PTH-mediated phosphorylation of Na+-K+-ATPase α1-subunit and PKCα without blocking ERK phosphorylation. Inhibition of Na+-K+-ATPase activity by the PLA2 metabolites arachidonic acid and 20-hydroxyeicosatetraenoic acid was prevented by specific inhibition of PKCα but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na+-K+-ATPase α1-subunit and PKCα, which was prevented by PLA2 inhibition. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by a sequential activation of a signaling pathway involving Src kinase, PLC, ERK, PLA2, and PKCα.
Intrarenal oxygenation: unique challenges and the biophysical basis of homeostasis
Authors: Evans Roger G. , Gardiner Bruce S. , Smith David W. , O'Connor Paul M.
Published: June 2008

The kidney is faced with unique challenges for oxygen regulation, both because its function requires that perfusion greatly exceeds that required to meet metabolic demand and because vascular control in the kidney is dominated by mechanisms that regulate glomerular filtration and tubular reabsorption. Because tubular sodium reabsorption accounts for most oxygen consumption (V̇o2) in the kidney, renal V̇o2varies with glomerular filtration rate. This provides an intrinsic mechanism to match changes in oxygen delivery due to changes in renal blood flow (RBF) with changes in oxygen demand. Renal V̇o2is low relative to supply of oxygen, but diffusional arterial-to-venous (AV) oxygen shunting provides a mechanism by which oxygen superfluous to metabolic demand can bypass the renal microcirculation. This mechanism prevents development of tissue hyperoxia and subsequent tissue oxidation that would otherwise result from the mismatch between renal V̇o2and RBF. Recent evidence suggests that RBF-dependent changes in AV oxygen shunting may also help maintain stable tissue oxygen tension when RBF changes within the physiological range. However, AV oxygen shunting also renders the kidney susceptible to hypoxia. Given that tissue hypoxia is a hallmark of both acute renal injury and chronic renal disease, understanding the causes of tissue hypoxia is of great clinical importance. The simplistic paradigm of oxygenation depending only on the balance between local perfusion and V̇o2is inadequate to achieve this goal. To fully understand the control of renal oxygenation, we must consider a triad of factors that regulate intrarenal oxygenation: local perfusion, local V̇o2, and AV oxygen shunting.
Quadriceps cross-sectional area changes in young healthy men with different magnitude of Q angle
Authors: Tsakoniti Aikaterini E. , Stoupis Christoforos A. , Athanasopoulos Spyros I.
Published: June 2008

Knee pain and dysfunction have been often associated with an ineffective pull of the patella by the vastus medialis (VM) relative to the vastus lateralis (VL), particularly in individuals with knee joint malalignment. Such changes in muscular behavior may be attributed to muscle inhibition and/or atrophy that precedes the onset of symptoms. The aim of this study was to investigate possible effects of knee joint malalignment, indicated by a high quadriceps (Q) angle (HQ angle >15°), on the anatomic cross-sectional area (aCSA) of the entire quadriceps and its individual parts, in a group of 17 young asymptomatic men compared with a group of 19 asymptomatic individuals with low Q angle (LQ angle <15°). The aCSA of the entire quadriceps (TQ), VM, VL, vastus intermedius (VI), rectus femoris (RF), and patellar tendon (PT) were measured during static and dynamic magnetic resonance imaging (MRI) with the quadriceps relaxed and under contraction, respectively. A statistically significant lower aCSA was obtained in the HQ angle group, compared with the LQ angle group, for the TQ, VL, and VI in both static (TQ = 9.9%, VL = 12.9%, and VI = 9.1%; P < 0.05) and dynamic imaging (TQ = 10.7%, P < 0.001; VL = 13.4%, P < 0.01; and VI = 9.8%, P < 0.05) and the aCSA of the VM in dynamic MRI (11.9%; P < 0.01). The muscle atrophy obtained in the HQ angle group may be the result of a protective mechanism that inhibits and progressively adapts muscle behavior to reduce abnormal loading and wear of joint structures.
Muscle fiber size and function in elderly humans: a longitudinal study
Authors: Frontera Walter R. , Reid Kieran F. , Phillips Edward M. , Krivickas Lisa S. , Hughes Virginia A. , Roubenoff Ronenn , Fielding Roger A.
Published: June 2008

Cross-sectional studies are likely to underestimate age-related changes in skeletal muscle strength and mass. The purpose of this longitudinal study was to assess whole muscle and single muscle fiber alterations in the same cohort of 12 older (mean age: start of study 71.1 ± 5.4 yr and end of study 80 ± 5.3 yr) volunteers (5 men) evaluated 8.9 yr apart. No significant changes were noted at follow-up in body weight, body mass index, and physical activity. Muscle strength, evaluated using isokinetic dynamometry, and whole muscle specific force of the knee extensors were significantly lower at follow-up. This was accompanied by a significant reduction (5.7%) in cross-sectional area of the total anterior muscle compartment of the thigh as evaluated by computed tomography. Muscle histochemistry showed no significant changes in fiber type distribution or fiber area. Experiments with chemically skinned single muscle fibers ( n = 411) demonstrated no change in type I fiber size but an increase in IIA fiber diameter. A trend toward an increase in maximal force in both fiber types was observed. Maximum unloaded shortening velocity did not change. In conclusion, single muscle fiber contractile function may be preserved in older humans in the presence of significant alterations at the whole muscle level. This suggests that surviving fibers compensate to partially correct muscle size deficits in an attempt to maintain optimal force-generating capacity.
Impaired neuromuscular function during isometric, shortening, and lengthening contractions after exercise-induced damage to elbow flexor muscles
Authors: Turner Tanya S. , Tucker Kylie J. , Rogasch Nigel C. , Semmler John G.
Published: June 2008

The purpose of this study was to examine the effect of exercise-induced damage of the elbow flexor muscles on steady motor performance during isometric, shortening, and lengthening contractions. Ten healthy individuals (age 22 ± 4 yr) performed four tasks with the elbow flexor muscles: a maximum voluntary contraction, a one repetition maximum (1 RM), an isometric task at three joint angles (short, intermediate, and long muscle lengths), and a constant-load task during slow (∼7°/s) shortening and lengthening contractions. Task performance was quantified as the fluctuations in wrist acceleration (steadiness), and electromyography was obtained from the biceps and triceps brachii muscles at loads of 10, 20, and 40% of 1 RM. Tasks were performed before, immediately after, and 24 h after eccentric exercise that resulted in indicators of muscle damage. Maximum voluntary contraction force and 1-RM load declined by ∼45% immediately after exercise and remained lower at 24 h (∼30% decrease). Eccentric exercise resulted in reduced steadiness and increased biceps and triceps brachii electromyography for all tasks. For the isometric task, steadiness was impaired at the short compared with the long muscle length immediately after exercise ( P < 0.01). Furthermore, despite no differences before exercise, there was reduced steadiness for the shortening compared with the lengthening contractions after exercise ( P = 0.01), and steadiness remained impaired for shortening contractions 24 h later ( P = 0.01). These findings suggest that there are profound effects for the performance of these types of fine motor tasks when recovering from a bout of eccentric exercise.
Cellular and Synaptic Actions of Acetylcholine in the Lamprey Spinal Cord
Authors: Quinlan Katharina A. , Buchanan James T.
Published: June 2008

This study investigated cellular and synaptic mechanisms of cholinergic neuromodulation in the in vitro lamprey spinal cord. Most spinal neurons tested responded to local application of acetylcholine (ACh) with depolarization and decreased input resistance. The depolarization persisted in the presence of either tetrodotoxin or muscarinic antagonist scopolamine and was abolished with nicotinic antagonist mecamylamine, indicating a direct depolarization through nicotinic ACh receptors. Local application of muscarinic ACh agonists modulated synaptic strength in the spinal cord by decreasing the amplitude of unitary excitatory and inhibitory postsynaptic potentials. The postsynaptic response to direct application of glutamate was unchanged by muscarinic agonists, suggesting a presynaptic mechanism. Cholinergic feedback from motoneurons was assessed using stimulation of a ventral root in the quiescent spinal cord while recording intracellularly from spinal motoneurons or interneurons. Mainly depolarizing potentials were observed, a portion of which was insensitive to removal of extracellular Ca2+, indicating electrotonic coupling. Hyperpolarizing potentials were also observed and were attenuated by the glycinergic antagonist strychnine, whereas depolarizing responses were potentiated by strychnine. Mecamylamine also reduced hyperpolarizing responses. The pharmacology of these responses suggests a Renshaw-like feedback pathway in lamprey. Immunohistochemistry for choline acetyltransferase, performed in combination with retrograde filling of motoneurons, demonstrated a population of nonmotoneuron cholinergic cells in the lamprey spinal cord. Thus endogenous cholinergic modulation of the lamprey spinal locomotor network is likely produced by both motoneurons and cholinergic interneurons acting via combined postsynaptic and presynaptic actions.
Altered Pontine Taste Processing in a Rat Model of Obesity
Authors: Kovacs Peter , Hajnal Andras
Published: June 2008

The increased palatability of modern diet contributes to eating beyond homeostatic need and in turn to the growing prevalence of obesity. How palatability is coded in taste-evoked neural activity and whether this activity differs between obese and lean remains unknown. To investigate this, we used extracellular single-unit recording in the second central gustatory relay, the pontine parabrachial nucleus while stimulating the tongue with various concentrations of sucrose (0.01–1.5 M) in Otsuka Long Evans Tokushima Fatty (OLETF) rats, lacking CCK-1R. The analyses included a total of 179 taste-responsive neurons in age-matched prediabetic, obese OLETF and lean Long Evans Tokushima Otsuka (LETO) controls. Compared with LETO, we found more NaCl-, and fewer sucrose-responsive neurons (67 vs. 47% and 14 vs. 32%), and an overall reduced response magnitude to sucrose in the OLETF rats. Further, in the obese rats there was a rightward shift in sucrose concentration-response functions relative to lean controls with a higher response-threshold (0.37 ± 0.05 vs. 0.23 ± 0.2 M, P < 0.05) and maximal neural response to higher sucrose concentrations (0.96 ± 0.07 vs. 0.56 ± 0.5 M, P < 0.001). These findings demonstrate altered central gustatory processing for sucrose in obese OLETF rat and further support the notion that palatability is encoded in the across neuron pattern.
Effects of Localized Intraspinal Injections of a Noradrenergic Blocker on Locomotion of High Decerebrate Cats
Authors: Delivet-Mongrain Hugo , Leblond Hugues , Rossignol Serge
Published: June 2008

Previous studies demonstrated that neuronal networks located in midlumbar segments (L3–L4) are critical for the expression of locomotion in cats following complete spinalization. In the present study the importance of several thoracolumbar segments (T8–L7) for the generation of spontaneous hindlimb locomotion in decerebrate cats was evaluated. Experiments were performed in high decerebrate cats ( n = 18) walking spontaneously. Yohimbine, an alpha2-noradrenergic antagonist, was microinjected intraspinally in various thoracolumbar segments. Locomotor performance was evaluated with kinematics and electromyographic (EMG) recordings before and after each injection. When and if spontaneous locomotion (SL) was abolished, skin or perineal stimuli (exteroceptive stimuli) were used to trigger locomotion (exteroceptive-induced locomotion [EL]). Yohimbine injections at L3 or L4 completely inhibited SL and EL. In contrast, injections at T8 did not interfere with SL or EL. Injections at T10, T11, T12, L5, L6, and L7 inhibited SL but EL could still be evoked. Injections at T13, L1, and L2 had similar effects except that the quality of locomotion evoked by exteroceptive stimulation declined. Combined injections at T13, L1, and L2 abolished SL and EL, in contrast to injections restricted to the same individual segments. Simultaneous injections at L5, L6, and L7 also abolished SL but EL could still be induced. These results suggest that noradrenergic mechanisms in L3–L4 segments are involved in the expression of locomotion in decerebrate cats, whereas antagonizing noradrenergic inputs in individual rostral or caudal segments may alter the expression and overall quality of the locomotor pattern without abolishing locomotion.
Characterization of Voltage-Dependent Ca2+Currents in IdentifiedDrosophilaMotoneurons In Situ
Authors: Worrell Jason W. , Levine Richard B.
Published: June 2008

Voltage-dependent Ca2+channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+channels in an intact system, but Ca2+currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+and K+channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+channels: Dmca1A, Dmca1D, and Dmα1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties.
Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies
Authors: Čolić Miodrag , Jovanović Suzana , Vasiljevski Milijana , Dujić Aleksandar
Published: June 2008

Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular
subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti‐CK
monoclonal antibodies (mAbs) using the streptavidin‐biotin immunoperoxidase method.
Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated
by double immunoflouorescence labeling. The obtained results showed that the expression
of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main
difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal
ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC,
respectively, whereas CK19 is mainly a pan‐TEC marker. In the adult animals, they are
localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and
some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC)
(CK10). The switch in their expression in the cortex was observed during the first two weeks
of postnatal life.
TCR V α‐ and V ß‐Gene Segment Use in T‐CellSubcultures Derived from a Type‐III Bare LymphocyteSyndrome Patient Deficient in MHC Class‐II Expression
Authors: Lambert M. , van Eggermond M. , Mascart F. , Dupont E. , van den Elsen P.
Published: June 2008

Previously, we and others have shown that MHC class‐II deficient humans have greatly
reduced numbers of CD4+CD8– peripheral T cells. These type‐III Bare Lymphocyte
Syndrome patients lack MHC class‐II and have an impaired MHC class‐I antigen
expression. In this study, we analyzed the impact of the MHC class‐II deficient
environment on the TCR V‐gene segment usage in this reduced CD4+CD8– T‐cell
subset. For these studies, we employed TcR V‐region‐specific monoclonal antibodies
(mAbs) and a semiquantitative PCR technique with V α and V ß amplimers, specific for
each of the most known V α‐ and V ß;‐gene region families. The results of our studies
demonstrate that some of the V α‐gene segments are used less frequent in the
CD4+CD8– T‐cell subset of the patient, whereas the majority of the TCR V α‐ and
V ß‐gene segments investigated were used with similar frequencies in both subsets in the
type‐III Bare Lymphocyte Syndrome patient compared to healthy control family
members. Interestingly, the frequency of TcR V α12 transcripts was greatly diminished
in the patient, both in the CD4+CD8– as well as in the CD4–CD8+ compartment,
whereas this gene segment could easily be detected in the healthy family controls. On
the basis of the results obtained in this study, it is concluded that within the reduced
CD4+CD8– T‐cell subset of this patient, most of the TCR V‐gene segments tested for are
employed. However, a skewing in the usage frequency of some of the V α‐gene segments
toward the CD4–CD8+ T‐cell subset was noticeable in the MHC class‐II deficient patient
that differed from those observed in the healthy family controls.
Susceptibility of Thymocytes for Infection by ChickenAnemia Virus is Related to Pre‐ and PosthatchingDevelopment
Authors: Jeurissen Suzan H. M. , Janse Marga E. , van Roozelaar Dirk J. , Koch Guus , de Boer Gerben F.
Published: June 2008

To investigate the age‐dependent mechanism of susceptibility for chicken anemia virus
(CAV) infection, we inoculated embryos and chickens of ages between day 9 of
embryonic development and day 28 after hatching with CAV. Chicken embryos
inoculated at days 9 and 11 of development showed no CAV‐infected cells in the
thymus, nor in other lymphoid organs. Many CAV‐infected cells were detected in the
thymic cortex of all chicken embryos inoculated at days 13 and 16 of development and
of all chickens inoculated 1, 3, and 7 days after hatching. All embryos and chickens that
contained CAV‐infected cells in the thymus also contained CAV‐infected cells in the
bone marrow, but not in the bursa of Fabricius or the spleen. In chickens inoculated at
days 14 and 21, only few CAV‐infected cells were detected in the thymus, whereas these
cells were not detected in thymi of 28‐day‐old inoculated chickens. Depletion of the
thymic cortex was only detected in chickens inoculated from day 16 of embryonic
development till day 21 after hatching. Only hematocrit values of the chickens
inoculated 1 and 3 days after hatching were below normal. The rationale for the
simultaneous susceptibility of cells of the T‐cell lineage and cells of the erythrocyte
lineage is discussed. As far as the thymus is concerned, the absence of clinical and
microscopical signs of CAV infection in older chickens and the inability of CAV to infect
embryos at days 9 and 11 of embryonic development may be caused by a lack of
susceptible thymocytes. In view of the three waves of thymic precursor cells that
populate the thymus during ontogeny, as described by Le Douarin and colleagues, we
hypothesize that CAV only infects thymocytes derived from the second wave of
precursor cells.
Human Thymic Epithelial Cells in Serum‐Free Culture: Nature andEffects on Thymocyte Cell Lines
Authors: Ropke Carsten , Elbroend Jette
Published: June 2008

Thymic epithelial cells (TEC) have been cultured for several months and/or for 4 to 5
transfers in a growth factor‐defined serum‐free medium without concurrent growth of
other cell types. The use of monoclonal antibodies and αMAM‐6 indicated that the
majority of TEC were of medullary origin. The vast majority of cells were positive for
LFA‐3 and class I, and class II expression, was low or absent. Supernatants from the
cultures were shown to contain IL‐1ß, IL‐6, and M‐CSF. Coculture of cloned
subpopulations of thymocytes and TEC showed effects of TEC and of secreted ILs on
thymocyte proliferation. High percentages of TEC were able to bind DN, DP, or SP
thymocyte populations, partly via CD2‐LFA‐3 adhesion. Thus, it is possible to culture
TEC without unknown serum factors and with maintenance of functional activities.