bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete Sequencing and Diversity Analysis of the Enterotoxin-Encoding Plasmids inClostridium perfringensType A Non-Food-Borne Human Gastrointestinal Disease Isolates
Authors: Miyamoto Kazuaki , Fisher Derek J. , Li Jihong , Sayeed Sameera , Akimoto Shigeru , McClane Bruce A.
Published: February 2006

Enterotoxin-producingClostridium perfringenstype A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carryingcpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lackingcpb2and IS1151but carryingcpeand IS1470-like sequences. By completely sequencing these twocpeplasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an ∼35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the ∼43.6-kb pCPF5603 variable region contains a functionalcpb2gene and several metabolic genes. Diversity studies indicated that other type A plasmidcpe+/IS1151SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmidcpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in thecpeplasmids of other type A SPOR/AAD isolates, as well as in representativeC. perfringenstype B to D isolates carrying other virulence plasmids, possibly suggesting that most or allC. perfringensvirulence plasmids transfer conjugatively.
Unusual Extra Space at the Active Site and High Activity for Acetylated Hydroxyproline of Prolyl Aminopeptidase from
Serratia marcescens
Authors: Nakajima Yoshitaka , Ito Kiyoshi , Sakata Makoto , Xu Yue , Nakashima Kanako , Matsubara Futoshi , Hatakeyama Susumi , Yoshimoto Tadashi
Published: February 2006

The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-
tert
-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-
tert
-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 Å resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-βNA (4-acetyloxyproline β-naphthylamide) was a better substrate than Pro-βNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.
The Crystal Structure of the Zinc Phosphodiesterase from
Escherichia coli
Provides Insight into Function and Cooperativity of tRNase Z-Family Proteins
Authors: Kostelecky Brenda , Pohl Ehmke , Vogel Andreas , Schilling Oliver , Meyer-Klaucke Wolfram
Published: February 2006

The
elaC
gene product from
Escherichia coli
, ZiPD, is a 3′ tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the
elaC
homologue from
Bacillus subtilis
,
E. coli elaC
is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3′ end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in
E. coli
are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 Å. This structure shares many features with the tRNase Z enzymes from
B. subtilis
and
Thermotoga maritima
, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the
B. subtilis
enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.
NnrA Is Required for Full Virulence and Regulates Several
Brucella melitensis
Denitrification Genes
Authors: Haine Valérie , Dozot Marie , Dornand Jacques , Letesson Jean-Jacques , De Bolle Xavier
Published: February 2006

We identified two regulators of denitrification genes in
Brucella melitensis
16M: NarR, which regulates the nitrate reductase (
nar
) operon, and NnrA, which is involved in the expression of the last three reductases of the denitrification pathway (
nirK
,
norB
, and
nosZ
). NnrA is required for virulence in mice and for intracellular resistance to nitric oxide.
Apyrase, the Product of the Virulence Plasmid-Encoded
phoN2
(
apy
) Gene of
Shigella flexneri
, Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread
Authors: Santapaola D. , Del Chierico F. , Petrucca A. , Uzzau S. , Casalino M. , Colonna B. , Sessa R. , Berlutti F. , Nicoletti M.
Published: February 2006

The role in virulence of the
Shigella flexneri ospB
-
phoN2
operon has been evaluated. Here we confirm that OspB is an effector and show that apyrase, the product of
phoN2
, may be a virulence factor, since it is required for efficient intercellular spreading. Apyrase may be important in a deoxynucleoside triphosphate-hydrolyzing activity-independent manner, suggesting that it may act as an interaction partner in the process of IcsA localization.
A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in
Streptococcus mutans
Authors: Hussain Haitham , Branny Pavel , Allan Elaine
Published: February 2006

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in
Streptococcus mutans
. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis.
Transcriptome Profiling of
Shewanella oneidensis
Gene Expression following Exposure to Acidic and Alkaline pH
Authors: Leaphart Adam B. , Thompson Dorothea K. , Huang Katherine , Alm Eric , Wan Xiu-Feng , Arkin Adam , Brown Steven D. , Wu Liyou , Yan Tingfen , Liu Xueduan , Wickham Gene S. , Zhou Jizhong
Published: February 2006

The molecular response of
Shewanella oneidensis
MR-1 to variations in extracellular pH was investigated based on genomewide gene expression profiling. Microarray analysis revealed that cells elicited both general and specific transcriptome responses when challenged with environmental acid (pH 4) or base (pH 10) conditions over a 60-min period. Global responses included the differential expression of genes functionally linked to amino acid metabolism, transcriptional regulation and signal transduction, transport, cell membrane structure, and oxidative stress protection. Response to acid stress included the elevated expression of genes encoding glycogen biosynthetic enzymes, phosphate transporters, and the RNA polymerase sigma-38 factor (
rpoS
), whereas the molecular response to alkaline pH was characterized by upregulation of
nhaA
and
nhaR
, which are predicted to encode an Na
+
/H
+
antiporter and transcriptional activator, respectively, as well as sulfate transport and sulfur metabolism genes. Collectively, these results suggest that
S. oneidensis
modulates multiple transporters, cell envelope components, and pathways of amino acid consumption and central intermediary metabolism as part of its transcriptome response to changing external pH conditions.
Global Transcriptome Analysis ofStaphylococcus aureusResponse to Hydrogen Peroxide
Authors: Chang Wook , Small David A. , Toghrol Freshteh , Bentley William E.
Published: February 2006

Staphylococcus aureusresponds with protective strategies against phagocyte-derived reactive oxidants to infect humans. Herein, we report the transcriptome analysis of the cellular response ofS. aureusto hydrogen peroxide-induced oxidative stress. The data indicate that the oxidative response includes the induction of genes involved in virulence, DNA repair, and notably, anaerobic metabolism.
MurQ Etherase Is Required by
Escherichia coli
in Order To Metabolize Anhydro-
N
-Acetylmuramic Acid Obtained either from the Environment or from Its Own Cell Wall
Authors: Uehara Tsuyoshi , Suefuji Kyoko , Jaeger Tina , Mayer Christoph , Park James T.
Published: February 2006

MurQ is an
N
-acetylmuramic acid-phosphate (MurNAc-P) etherase that converts MurNAc-P to
N
-acetylglucosamine-phosphate and is essential for growth on MurNAc as the sole source of carbon (T. Jaegar, M. Arsic, and C. Mayer, J. Biol. Chem.
280:
30100-30106, 2005). Here we show that MurQ is the only MurNAc-P etherase in
Escherichia coli
and that MurQ and AnmK kinase are required for utilization of anhydro-MurNAc derived either from cell wall murein or imported from the medium.
Repressor of Phage
16
-
3
with Altered Binding Specificity Indicates Spatial Differences in Repressor-Operator Complexes
Authors: Ferenczi Szilamér , Orosz László , Papp Péter P.
Published: February 2006

The C repressor protein of phage
16-3
, which is required for establishing and maintaining lysogeny, recognizes structurally different operators which differ by 2 bp in the length of the spacer between the conserved palindromic sequences. A “rotationally flexible protein homodimers” model has been proposed in order to explain the conformational adaptivity of the
16-3
repressor. In this paper, we report on the isolation of a repressor mutant with altered binding specificity which was used to identify a residue-base pair contact and to monitor the spatial relationship of the recognition helix of C repressor to the contacting major groove of DNA within the two kinds of repressor-operator complexes. Our results indicate spatial differences at the interface which may reflect different docking arrangements in recognition of the structurally different operators by the
16-3
repressor.
PLANT UNCOUPLING MITOCHONDRIAL PROTEINS
Authors: Vercesi Aníbal Eugênio , Borecký Jiri , Maia Ivan de Godoy , Arruda Paulo , Cuccovia Iolanda Midea , Chaimovich Hernan
Published: February 2006

Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide-sensitive free fatty acid-activated H+ flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (ΔμH+) and production of reactive oxygen species.
MOSSES AS MODEL SYSTEMS FOR THE STUDY OF METABOLISM AND DEVELOPMENT
Authors: Cove David , Bezanilla Magdalena , Harries Phillip , Quatrano Ralph
Published: February 2006

The haploid gametophyte stage of the moss life cycle is amenable to genetic and biochemical studies. Many species can be cultured on simple defined media, where growth is rapid, making them ideal material for metabolic studies. Developmental responses to hormones and to environmental inputs can be studied both at the level of individual cells and in multicellular tissues. The protonemal stage of gametophyte development comprises cell filaments that extend by the serial division of their apical cells, allowing the investigation of the generation and modification of cell polarity and the role of the cytoskeleton in these processes. Molecular techniques including gene inactivation by targeted gene replacement or by RNA interference, together with the nearly completed sequencing of the Physcomitrella patens genome, open the way for detailed study of the functions of genes involved in both development and metabolism.
Relations Between Structure and Function of the Mitochondrial ADP/ATP Carrier
Authors: Nury H. , Dahout-Gonzalez C. , Trézéguet V. , Lauquin G.J.M. , Brandolin G. , Pebay-Peyroula E.
Published: February 2006

Import and export of metabolites through mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner membrane. Proteins of the mitochondrial carrier family ( MCF ) are embedded in this membrane, and each member of the family achieves the selective transport of a specific metabolite. Among these, the ADP/ATP carrier transports ADP into the mitochondrial matrix and exports ATP toward the cytosol after its synthesis. Because of its natural abundance, the ADP/ATP carrier is the best characterized within MCF, and a high-resolution structure of one conformation is known. The overall structure is basket shaped and formed by six transmembrane helices that are not only tilted with respect to the membrane, but three of them are also kinked at the level of prolines. The functional mechanisms, nucleotide recognition, and conformational changes for the transport, suggested from the structure, are discussed along with the large body of biochemical and functional results.
Pathogenic role of Fgf23 in Hyp mice
Authors: Liu Shiguang , Zhou Jianping , Tang Wen , Jiang Xi , Rowe David W. , Quarles L. Darryl
Published: February 2006

Inactivating mutations of the PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) endopeptidase, the disease-causing gene in X-linked hypophosphatemia (XLH), results in increased circulating levels of fibroblastic growth factor-23 (FGF23), a bone-derived phosphaturic factor. To determine the causal role of FGF23 in XLH, we generated a combined Fgf23-deficient enhanced green fluorescent protein (eGFP) reporter and Phex-deficient Hyp mouse model ( Fgf23+/−/ Hyp). eGFP expression was expressed in osteocytes embedded in bone that exhibited marked upregulation of eGFP in response to Phex deficiency and in CD31-positive cells in bone marrow venules that expressed low eGFP levels independently of Phex. In bone marrow stromal cells (BMSCs) derived from Fgf23−/−/ Hyp mice, eGFP expression was also selectively increased in osteocyte-like cells within mineralization nodules and detected in low levels in CD31-positive cells. Surprisingly, eGFP expression was not increased in cell surface osteoblasts, indicating that Phex deficiency is necessary but not sufficient for increased Fgf23 expression in the osteoblast lineage. Additional factors, associated with either osteocyte differentiation and/or extracellular matrix, are necessary for Phex deficiency to stimulate Fgf23 gene transcription in bone. Regardless, the deletion of Fgf23 from Hyp mice reversed the hypophosphatemia, abnormal 1,25(OH)2D3 levels, rickets, and osteomalacia associated with Phex deficiency. These results suggest that Fgf23 acts downstream of Phex to cause both the renal and bone phenotypes in Hyp mice.
Acute, same-day effects of antecedent exercise on counterregulatory responses to subsequent hypoglycemia in type 1 diabetes mellitus
Authors: Sandoval Darleen A. , Aftab Guy Deanna L. , Richardson M. Antoinette , Ertl Andrew C. , Davis Stephen N.
Published: February 2006

Exercise-induced hypoglycemia can occur within hours after exercise in type 1 diabetes mellitus (T1DM) patients. This study tested the hypothesis that an acute exercise bout causes (within hours) blunted autonomic and metabolic responses to subsequent hypoglycemia in patients with T1DM. Twelve T1DM patients (3 W/9 M) were studied during a single-step, 2-h hyperinsulinemic (572 ± 4 pmol/l) hypoglycemic (2.8 ± 0.1 mmol/l) clamp 2 h after either a hyperinsulinemic euglycemic (AM EUG) or hypoglycemic clamp (AM HYPO) or after sitting in a chair with basal insulin infusion (AM CON) or 90 min of moderate-intensity exercise (50% V̇o2 max, AM EX). Both AM HYPO and AM EX significantly blunted epinephrine responses and muscle sympathetic nerve activity responses to subsequent hypoglycemia compared with both control groups. Endogenous glucose production was significantly lower and the exogenous glucose infusion rate needed to maintain the hypoglycemic level was significantly greater during subsequent hypoglycemia in AM EX vs. CON. Rate of glucose disposal (Rd) was significantly reduced following AM HYPO. In summary, within 2.5 h, both moderate-intensity AM EX and AM HYPO blunted key autonomic counterregulatory responses. Despite this, glucose Rdwas reduced during afternoon hypoglycemia following morning hypoglycemia, indicating posthypoglycemic insulin resistance. After morning exercise, endogenous glucose production was blunted, but glucose Rdwas maintained during afternoon hypoglycemia, thereby indicating reduced metabolic defenses against hypoglycemia. These data suggest that exercise-induced counterregulatory failure can occur very rapidly, increasing the risk for hypoglycemia in T1DM within hours.
Perturbed skeletal muscle insulin signaling in the adult female intrauterine growth-restricted rat
Authors: Oak Shilpa A. , Tran Cang , Pan Gerald , Thamotharan Mannikkavasagar , Devaskar Sherin U.
Published: February 2006

To determine the molecular mechanism(s) linking fetal adaptations in intrauterine growth restriction (IUGR) to adult maladaptations of type 2 diabetes mellitus, we investigated the effect of prenatal seminutrient restriction, modified by early postnatal ad libitum access to nutrients (CM/SP) or seminutrient restriction (SM/SP), vs. early postnatal seminutrient restriction alone (SM/CP) or control nutrition (CM/CP) on the skeletal muscle postreceptor insulin-signaling pathway in the adult offspring. The altered in utero hormonal/metabolic milieu was associated with no change in basal total IRS-1, p85, and p110β subunits of PI 3-kinase, PKCθ, and PKCζ concentrations but an increase in basal IRS-2 ( P < 0.05) only in the CM/SP group and an increase in basal phospho (p)-PDK-1 ( P < 0.05), p-Akt ( P < 0.05), and p-PKCζ ( P < 0.05) concentrations in the CM/SP and SM/SP groups. Insulin-stimulated increases in p-PDK-1 ( P < 0.05) and p-Akt ( P < 0.0007), with no increase in p-PKCζ, were seen in both CM/SP and SM/SP groups. SHP2 ( P < 0.03) and PTP1B ( P < 0.03) increased only in SM/SP with no change in PTEN in CM/SP and SM/SP groups. Aberrations in kinase and phosphatase moieties in the adult IUGR offspring were initiated in utero but further sculpted by the early postnatal nutritional state. Although the CM/SP group demonstrated enhanced kinase activation, the SM/SP group revealed an added increase in phosphatase concentrations with the net result of heightened basal insulin sensitivity in both groups. The inability to further respond to exogenous insulin was due to the key molecular distal roadblock consisting of resistance to phosphorylate and activate PKCζ necessary for GLUT4 translocation. This protective adaptation may become maladaptive and serve as a forerunner for gestational and type 2 diabetes mellitus.
Computational model of in vivo human energy metabolism during semistarvation and refeeding
Authors: Hall Kevin D.
Published: February 2006

Changes in body weight and composition are the result of complex interactions among metabolic fluxes contributing to macronutrient balances. To better understand these interactions, a mathematical model was constructed that used the measured dietary macronutrient intake during semistarvation and refeeding as model inputs and computed whole body energy expenditure, de novo lipogenesis, and gluconeogenesis as well as turnover and oxidation of carbohydrate, fat, and protein. Published in vivo human data provided the basis for the model components that were integrated by fitting a few unknown parameters to the classic Minnesota human starvation experiment. The model simulated the measured body weight and fat mass changes during semistarvation and refeeding and predicted the unmeasured metabolic fluxes underlying the body composition changes. The resting metabolic rate matched the experimental measurements and required a model of adaptive thermogenesis. Refeeding caused an elevation of de novo lipogenesis that, along with increased fat intake, resulted in a rapid repletion and overshoot of body fat. By continuing the computer simulation with the prestarvation diet and physical activity, the original body weight and composition were eventually restored, but body fat mass was predicted to take more than one additional year to return to within 5% of its original value. The model was validated by simulating a recently published short-term caloric restriction experiment without changing the model parameters. The predicted changes in body weight, fat mass, resting metabolic rate, and nitrogen balance matched the experimental measurements, thereby providing support for the validity of the model.
Glucocorticoid-deficient corticotropin-releasing hormone knockout mice maintain glucose requirements but not autonomic responses during repeated hypoglycemia
Authors: Jacobson Lauren , Ansari Tasneem , Potts Jessica , McGuinness Owen P.
Published: February 2006

Glucocorticoids have been implicated in hypoglycemia-induced autonomic failure but also contribute to normal counterregulation. To determine the influence of normal and hypoglycemia-induced levels of glucocorticoids on counterregulatory responses to acute and repeated hypoglycemia, we compared plasma catecholamines, corticosterone, glucagon, and glucose requirements in male wild-type (WT) and glucocorticoid-deficient, corticotropin-releasing hormone knockout (CRH KO) mice. Conscious, chronically cannulated, unrestrained WT and CRH KO mice underwent a euglycemic (Prior Eu) or hypoglycemic clamp (Prior Hypo) on day 1 followed by a hypoglycemic clamp on day 2 (blood glucose both days, 65 ± 1 mg/dl). Baseline epinephrine and glucagon were similar, and norepinephrine was elevated, in CRH KO vs. WT mice. CRH KO corticosterone was almost undetectable (<1.5 μg/dl) and unresponsive to hypoglycemia. CRH KO glucose requirements were significantly higher during day 1 hypoglycemia despite epinephrine and glucagon responses that were comparable to or greater than those in WT. Hyperinsulinemic euglycemia did not increase hormones or glucose requirements above baseline. On day 2, Prior Hypo WT had significantly higher glucose requirements and significantly lower corticosterone and glucagon responses. Prior Hypo and Prior Eu CRH KO mice had similar day 2 glucose requirements. However, Prior Hypo CRH KO mice had significantly lower day 2 epinephrine and norepinephrine vs. Prior Eu CRH KO and tended to have lower glucagon than on day 1. We conclude that glucocorticoid insufficiency in CRH KO mice correlates with 1) impaired counterregulation during acute hypoglycemia and 2) complex effects after repeated hypoglycemia, neither preventing decreased hormone responses nor worsening glucose requirements.
Exercise training and calorie restriction increase SREBP-1 expression and intramuscular triglyceride in skeletal muscle
Authors: Nadeau Kristen J. , Ehlers Lindsay B. , Aguirre Lina E. , Moore Russell L. , Jew Korinne N. , Ortmeyer Heidi K. , Hansen Barbara C. , Reusch Jane E. B. , Draznin Boris
Published: February 2006

Intramuscular triglyceride (IMTG) deposition in skeletal muscle is associated with obesity and type 2 diabetes (T2DM) and is thought to be related to insulin resistance (IR). Curiously, despite enhanced skeletal muscle insulin sensitivity, highly trained athletes and calorie-restricted (CR) monkeys also have increased IMTG. Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the biosynthesis of cholesterol and fatty acids. SREBP-1 is increased by insulin in skeletal muscle in vitro and in skeletal muscle of IR subjects, but SREBP-1 expression has not been examined in exercise training or calorie restriction. We examined the relationship between IMTG and SREBP-1 expression in animal models of exercise and calorie restriction. Gastrocnemius and soleus muscle biopsies were obtained from 38 Sprague-Dawley rats (18 control and 20 exercise trained). Triglyceride content was higher in the gastrocnemius and soleus muscles of the trained rats. SREBP-1c mRNA, SREBP-1 precursor and mature proteins, and fatty acid synthase (FAS) protein were increased with exercise training. Monkeys ( Macaca mulatta) were CR for a mean of 10.4 years, preventing weight gain and IR. Vastus lateralis muscle was obtained from 12 monkeys (6 CR and 6 controls). SREBP-1 precursor and mature proteins and FAS protein were higher in the CR monkeys. In addition, phosphorylation of ERK1/ERK2 was increased in skeletal muscle of CR animals. In summary, SREBP-1 protein and SREBP-1c mRNA are increased in interventions that increase IMTG despite enhanced insulin sensitivity. CR and exercise-induced augmentation of SREBP-1 expression may be responsible for the increased IMTG seen in skeletal muscle of highly conditioned athletes.
Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function
Authors: Shao Ruijin , Weijdegård Birgitta , Ljungström Karin , Friberg Anders , Zhu Changlian , Wang Xiaoyang , Zhu Yihong , Fernandez-Rodriguez Julia , Egecioglu Emil , Rung Emilia , Billig Håkan
Published: February 2006

Progesterone and its interaction with nuclear progesterone receptors (PR) PR-A and PR-B play a critical role in the regulation of female reproductive function in all mammals. However, our knowledge of the regulation and possible cellular function of PR protein isoforms in the fallopian tube and uterus in vivo is still very limited. In the present study, we revealed that equine chorionic gonadotropin (eCG) treatment resulted in a time-dependent increase in expression of both isoforms, reaching a maximal level at 48 h in the fallopian tube. Regulation of PR-A protein expression paralleled that of PR-B protein expression. However, in the uterus PR-B protein levels increased and peaked earlier than PR-A protein levels after eCG treatment. With prolonged exposure to eCG, PR-B protein levels decreased, whereas PR-A protein levels continued to increase. Furthermore, subsequent treatment with human (h)CG decreased the levels of PR protein isoforms in both tissues in parallel with increased endogenous serum progesterone levels. To further elucidate whether progesterone regulates PR protein isoforms, we demonstrated that a time-dependent treatment with progesterone (P4) decreased the expression of PR protein isoforms in both tissues, whereas decreases in p27, cyclin D2, and proliferating cell nuclear antigen protein levels were observed only in the uterus. To define the potential PR-mediated effects on apoptosis, we demonstrated that the PR antagonist treatment increased the levels of PR protein isoforms, induced mitochondrial-associated apoptosis, and decreased in epidermal growth factor (EGF) and EGF receptor protein expression in both tissues. Interestingly, immunohistochemistry indicated that the induction of apoptosis by PR antagonists was predominant in the epithelium, whereas increase in PR protein expression was observed in stromal cells of both tissues. Taken together, these observations suggest that 1) the tissue-specific and hormonal regulation of PR isoform expression in mouse fallopian tube and uterus, where they are potentially involved in regulation of mitochondrial-mediated apoptosis depending on the cellular compartment; and 2) a possible interaction between functional PR protein and growth factor signaling may have a coordinated role for regulating apoptotic process in both tissues in vivo.
Activation of the Reg family genes by pancreatic-specific IGF-I gene deficiency and after streptozotocin-induced diabetes in mouse pancreas
Authors: Lu Yarong , Ponton André , Okamoto Hiroshi , Takasawa Shin , Herrera Pedro L. , Liu Jun-Li
Published: February 2006

We have recently reported that Pdx1-Cre-mediated whole pancreas inactivation of IGF-I gene [in pancreatic-specific IGF-I gene-deficient (PID) mice] results in increased β-cell mass and significant protection against both type 1 and type 2 diabetes. Because the phenotype is unlikely a direct consequence of IGF-I deficiency, the present study was designed to explore possible activation of proislet factors in PID mice by using a whole genome DNA microarray. As a result, multiple members of the Reg family genes (Reg2, -3α, and -3β, previously not known to promote islet cell growth) were significantly upregulated in the pancreas. This finding was subsequently confirmed by Northern blot and/or real-time PCR, which exhibited 2- to 8-fold increases in the levels of these mRNAs. Interestingly, these Reg family genes were also activated after streptozotocin-induced β-cell damage and diabetes (wild-type T1D mice) when islet cells were undergoing regeneration. Immunohistochemistry revealed increased Reg proteins in exocrine as well as endocrine pancreas and suggested their potential role in β-cell neogenesis in PID or T1D mice. Previously, other Reg proteins (Reg1 and islet neogenesis-associated protein) have been shown to promote islet cell replication and neogenesis. These uncharacterized Reg proteins may play a similar but more potent role, not only in normal islet cell growth in PID mice, but also in islet cell regeneration after T1D.
cGMP stimulates renin secretion in vivo by inhibiting phosphodiesterase-3
Authors: Beierwaltes William H.
Published: February 2006

The interaction between renin, nitric oxide (NO), and its second messenger cGMP is controversial. cAMP is the stimulatory second messenger for renin but is degraded by phosphodiesterases (PDEs). We previously reported that increasing endogenous cGMP in rats by inhibiting its breakdown by PDE-5 stimulated renin secretion rate (RSR). This could be reversed by selective inhibition of neuronal nitric oxide synthase (nNOS). PDE-3 metabolizes cAMP, but this can be inhibited by cGMP, suggesting that renal cGMP could stimulate RSR by diminishing PDE-3 degradation of cAMP. Rats were anesthetized with Inactin before determination of blood pressure (BP), renal blood flow (RBF), and sampling of renal venous and arterial blood to determine RSR. In 13 rats, basal BP was 104 ± 2 mmHg, RBF was 6.1 ml·min−1·g kidney wt−1and RSR was 2.9 ± 1.4 ng ANG I·h−1·min−1. Inhibiting PDE-5 with 20 mg/kg body wt ip Zaprinast did not change hemodynamic parameters but increased RSR fivefold to 12.2 ± 4.9 ng ANG I·h−1·min−1( P < 0.05). Renal venous cAMP was increased by Zaprinast from 93.8 ± 27.9 to 149.2 ± 36.0 pM·min−1·g kidney wt−1( P < 0.05). When another 10 rats were treated with the PDE-3 inhibitor Milrinone (0.4 μg/min over 30 min, which did not affect hemodynamics), RSR was elevated to 10.4 ± 4.4 ng ANG I·h−1·min−1. Milrinone also increased renal venous cAMP from 212 ± 29 to 304 ± 29 pM·min−1·g kidney wt−1( P < 0.025). Administration of Zaprinast to rats pretreated with Milrinone ( n = 10) did not further increase in RSR (7.5 ± 3.3 ng ANG I·h−1·min−1). These results are consistent with endogenous renal cGMP inhibiting PDE-3, which diminishes renal metabolism of cAMP. The resulting increase in cAMP serves as an endogenous stimulus for renin secretion. This suggests a pathway by which NO can indirectly stimulate RSR through its second messenger cGMP.
Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes
Authors: Abouhamed Marouan , Gburek Jakub , Liu Wei , Torchalski Blazej , Wilhelm Andreas , Wolff Natascha A. , Christensen Erik I. , Thévenod Frank , Smith Craig P.
Published: February 2006

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.
Urea flux across MDCK-mUT-A2 monolayers is acutely sensitive to AVP, cAMP, and [Ca2+]i
Authors: Potter Elizabeth A. , Stewart Gavin , Smith Craig P.
Published: February 2006

In this study, we engineered a Madin-Darby canine kidney (MDCK) type I cell line to stably express the mouse urea transporter UT-A2. Monolayers of MDCK-mUT-A2 cells had a basal phloretin-inhibitable urea permeability of 8.4 × 10−6± 0.3 cm/s. Treatment of MDCK-mUT-A2 monolayers with AVP led to a rapid dose-dependent increase in trans-monolayer phloretin-inhibitable urea flux. The temporal pattern of response was markedly different from that observed for MDCK cells expressing rat UT-A1. Exposure of MDCK-mUT-A2 cells to either 10 μM forskolin or 250 μM 8-bromo cAMP also increased urea flux rate. Inclusion of the PKA inhibitor H89 (10 μM) had no effect on the forskolin-stimulated increase in urea flux across MDCK-mUT-A2 monolayers. Treatment with either 10 μM CPA or 1 mM ATP also caused an increase in UT-A2-mediated urea flux, although these responses where transient compared with those induced by AVP or elevated cAMP. Taken together, these results show for the first time that UT-A2 is acutely sensitive to AVP, cAMP, or increased intracellular calcium.
Overexpression of kidney neutral endopeptidase (EC 3.4.24.11) and renal function in experimental cirrhosis
Authors: Sansoè G. , Aragno M. , Mastrocola R. , Cutrin J. C. , Silvano S. , Mengozzi G. , Smedile A. , Rosina F. , Danni O. , Rizzetto M.
Published: February 2006

Neutral endopeptidase degrades atrial natriuretic peptide (ANP) and bradykinin and may generate endothelin-1 from big-endothelin. In advanced cirrhosis, sodium retention is accompanied by elevated plasma ANP levels, and infusion of ANP causes hypotension, but in normal humans increasing the concentration of ANP through the inhibition of neutral endopeptidase, localized in renal proximal tubule cells, causes natriuresis without any arterial pressure drop. The purpose of this study was the assessment of kidney neutral endopeptidase expression and responses to candoxatrilat (a specific inhibitor of this enzyme) in rats with CCl4-induced cirrhosis. Two groups of control rats ( n = 5) were injected with vehicle or 3 mg/kg candoxatrilat. Three groups of cirrhotic rats with ascites ( n = 10) received vehicle alone or 3 or 10 mg/kg candoxatrilat. In cirrhotic rats, Western blot analysis revealed a 170% increase in renal neutral endopeptidase protein content ( P < 0.03), mainly in the proximal nephron and macula densa, and both candoxatrilat dosages increased plasma ANP levels, urinary volume, and urinary excretion of sodium, ANP, and cGMP compared with vehicle alone (all P < 0.03). Candoxatrilat (10 mg/kg) also reduced tubular solute-free water reabsorption ( P < 0.03) in cirrhotic rats, but renal blood flow, arterial pressure, and plasma renin activity were unaffected. Neutral endopeptidase inhibition has natriuretic and aquaretic actions in cirrhosis without any effect on blood pressure and kidney perfusion due to a significant overexpression of this enzyme in renal cortex.
Methyl-β-cyclodextrin induces vasopressin-independent apical accumulation of aquaporin-2 in the isolated, perfused rat kidney
Authors: Russo Leileata M. , McKee Mary , Brown Dennis
Published: February 2006

Vasopressin increases urine concentration by stimulating plasma membrane accumulation of aquaporin-2 (AQP2) in collecting duct principal cells, allowing bulk water flow across the collecting duct from lumen to interstitium down an osmotic gradient. Mutations in the vasopressin type 2 receptor (V2R) cause hereditary X-linked nephrogenic diabetes insipidus (NDI), a disease characterized by excessive urination and dehydration. Recently, we showed that inhibition of endocytosis by the cholesterol-depleting drug methyl-β-cyclodextrin (mβCD) induces plasma membrane accumulation of AQP2 in transfected renal epithelial cells overexpressing epitope-tagged AQP2. Here, we asked whether mβCD could induce membrane accumulation of AQP2 in situ using the isolated, perfused kidney (IPK). By immunofluorescence and electron microscopy, we show that AQP2 was shifted from a predominantly intracellular localization to the apical membrane of principal cells following 1-h perfusion of Sprague-Dawley rat kidneys with 5 mM mβCD. Quantification of staining revealed that the intensity of AQP2 was increased from 647 ± 114 (control) to 1,968 ± 299 units (mβCD; P < 0.001), an effect similar to that seen after perfusion with 4 nM dDAVP (1,860 ± 298, P < 0.001). Similar changes were observed following mβCD perfusion of kidneys from vasopressin-deficient Brattleboro rats. No effect of mβCD treatment on the basolateral distribution of AQP3 and AQP4 was detected. These data indicate that AQP2 constitutively recycles between the apical membrane and intracellular vesicles in principal cells in situ and that inducing apical AQP2 accumulation by inhibiting AQP2 endocytosis is a feasible goal for bypassing the defective V2R signaling pathway in X-linked NDI.
Furin cleavage activates the epithelial Na+channel by relieving Na+self-inhibition
Authors: Sheng Shaohu , Carattino Marcelo D. , Bruns James B. , Hughey Rebecca P. , Kleyman Thomas R.
Published: February 2006

Epithelial Na+channels (ENaC) are inhibited by extracellular Na+, a process referred to as Na+self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in α, or one site in γ, blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111–18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant α-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+self-inhibition response, while channels with a mutant γ-subunit showed a modestly enhanced Na+self-inhibition response. Analysis of Na+self-inhibition at varying [Na+] indicates that channels containing mutant α-subunits exhibit an increased Na+affinity. At the single-channel level, channels with a mutant α-subunit had a low open probability ( Po) in the presence of a high external [Na+] in the patch pipette. Podramatically increased when trypsin was also present, or when a low external [Na+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na+self-inhibition and that activation requires that the α-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC Poand extracellular [Na+], supporting the notion that Na+self-inhibition reflects a Poreduction due to high extracellular [Na+].
Hippocampal Network Hyperactivity After Selective Reduction of Tonic Inhibition in GABAA Receptor α5 Subunit–Deficient Mice
Authors: Glykys Joseph , Mody Istvan
Published: February 2006

Functionally, γ-aminobutyric acid receptor (GABAR)–mediated inhibition can be classified as phasic (synaptic) and tonic (extrasynaptic). The GABARs underlying tonic inhibition assemble from subunits different from those responsible for phasic inhibition. We wanted to assess the excitability of hippocampal pyramidal cell (PC) networks following a selective impairment of tonic inhibition. This is difficult to accomplish by pharmacological means. Because the GABAR α5 subunits mostly mediate the tonic inhibition in CA1 and CA3 PCs, we quantified changes in tonic inhibition and examined network excitability in slices of adult gabra5−/− mice. In gabra5−/− CA1 and CA3 PCs tonic inhibitory currents were 60 and 53%, respectively, of those recorded in wild type (WT), with no alterations in phasic inhibition. The amount of tonic inhibition recorded in slices was significantly affected by the method of slice storage (interface or submerged chamber). Field recordings in gabra5−/− CA3 pyramidal layer showed an increased network excitability that was decreased by the GABAR agonist muscimol at a concentration that restored the tonic inhibition of gabra5−/− PCs to the WT level without altering phasic inhibition. Through a battery of pharmacological experiments, we have identified δ subunit–containing GABARs as the mediators of the residual tonic inhibition in gabra5−/− PCs. Our study is consistent with an important role of tonic inhibition in the control of hippocampal network excitability and highlights selective enhancers of tonic inhibition as promising therapeutic approaches for diseases involving network hyperexcitability.
Metabotropic Receptor-Dependent Long-Term Depression Persists in the Absence of Protein Synthesis in the Mouse Model of Fragile X Syndrome
Authors: Nosyreva Elena D. , Huber Kimberly M.
Published: February 2006

Fragile X syndrome (FXS), a form of human mental retardation, is caused by loss of function mutations in the fragile X mental retardation gene ( FMR1). The protein product of FMR1, fragile X mental retardation protein (FMRP) is an RNA-binding protein and may function as a translational suppressor. Metabotropic glutamate receptor–dependent long-term depression (mGluR-LTD) in hippocampal area CA1 is a form of synaptic plasticity that relies on dendritic protein synthesis. mGluR-LTD is enhanced in the mouse model of FXS, Fmr1 knockout (KO) mice, suggesting that FMRP negatively regulates translation of proteins required for LTD. Here we examine the synaptic and cellular mechanisms of mGluR-LTD in KO mice and find that mGluR-LTD no longer requires new protein synthesis, in contrast to wild-type (WT) mice. We further show that mGluR-LTD in KO and WT mice is associated with decreases in AMPA receptor (AMPAR) surface expression, indicating a similar postsynaptic expression mechanism. However, like LTD, mGluR-induced decreases in AMPAR surface expression in KO mice persist in protein synthesis inhibitors. These results are consistent with recent findings of elevated protein synthesis rates and synaptic protein levels in Fmr1 KO mice and suggest that these elevated levels of synaptic proteins are available to increase the persistence of LTD without de novo protein synthesis.
Mappingcis-acting regulatory variation in recombinant congenic strains
Authors: Lee Peter D. , Ge Bing , Greenwood Celia M. T. , Sinnett Donna , Fortin Yannick , Brunet Sebastien , Fortin Anny , Takane Marina , Skamene Emil , Pastinen Tomi , Hallett Michael , Hudson Thomas J. , Sladek Robert
Published: February 2006

We present an integrated approach for the enriched detection of genes subject to cis-acting variation in the mouse genome. Gene expression profiling was performed with lung tissue from a panel of recombinant congenic strains (RCS) derived from A/J and C57BL/6J inbred mouse strains. A multiple-regression model measuring the association between gene expression level, donor strain of origin (DSO), and predominant strain background identified over 1,500 genes ( P < 0.05) whose expression profiles differed according to the DSO. This model also identified over 1,200 genes whose expression showed dependence on background ( P < 0.05), indicating the influence of background genetic context on transcription levels. Sequences obtained from 1-kb segments of 3′-untranslated regions identified single nucleotide polymorphisms in 64% of genes whose expression levels correlated with DSO status, compared with 29% of genes that displayed no association ( P < 0.01, Fisher exact test). Allelic imbalance was identified in 50% of genes positive for expression-DSO association, compared with 22% of negative genes ( P < 0.05, Fisher exact test). Together, these results demonstrate the utility of RCS mice for identifying the roles of proximal genetic determinants and background genetic context in determining gene expression levels. We propose the use of this integrated experimental approach in multiple tissues from this and other RCS panels as a means for genome-wide cataloging of genetic regulatory mechanisms in laboratory strains of mice.