bluuebunny/crossref_metadata_2025_split huggingface dataset:
HLA A2 restricted HIV specific mucosal and systemic immunity induced with secreted heat shock protein gp96‐Ig
Authors: Strbo Natasa , Pahwa Savita , Kolber Michael , Fisher Eva , Franchini Genoveffa , Podack Eckhard 
Published: June 2021

Heat shock protein gp96 is the primary chaperone of the endoplasmic reticulum and a natural adjuvant for priming the innate and adaptive immune system. We have generated a secreted gp96‐fusion protein (gp96‐Ig) that mediated strong, antigen specific CD8‐CTL expansion in vivo.The aim of this study was to generate and test gp96‐Ig secreting and HIV‐antigen expressing cells as vaccines for HIV.Murine NIH‐3T3 or human 293 cells were co‐transfected with gp96‐Ig and HIVgag‐gfp cDNAs. To ensure high level of expression of HIV antigen transfected cells were sorted for gag‐gfp+ cells and level of gp96‐Ig secretion was determined by ELISA. We used H2Dd‐HLA A2 transgenic mice and immunize them with 3T3‐HIV‐gag‐gfp‐gp96‐Ig. The frequency of the murine gag‐specific CD8 CTL response was quantified with HLA A2 tetramers loaded with HIV‐gag‐peptide (SLYNTVATL). HLA A2 tetramer specific CD8 cells were found in the peripheral blood, spleen, peritoneal cavity as well as at mucosal sites (Peyer's Patches, intraepithelial compartment and lamina propria) within five days after intraperitoneal injection of 3T3‐HIV‐gag‐gfp‐gp96‐Ig.Our results imply that allogeneic cells secreting gp96‐Ig with associated HIV gag‐peptides work well as vaccines and are inducing both mucosal and systemic immunity.This study was supported by NIH/NIAID grants AI073234 and AI068515
Leucine oxidation in its excess in growing rats: Proposal of the method to determine safe intake level for leucine
Authors: Matsumoto Hideki , Kodama Riho , Amao Michiko , Miura Makoto , Kimura Takeshi , Sakai Ryosei 
Published: June 2021

Animals have ability to adapt to various dietary intake levels of protein and amino acids (AA) by controlling enzymatic activities. Therefore, we hypothesized that leucine (Leu) intakes within its catabolic capacity are safe levels, and investigated Leu‐oxidizing capacity in growing rats.Male F344 rats (N=88) were accustomed to 9h of feeding of diets containing graded doses of additional Leu (0–30%) for 14 days. After 15h of fasting period, rats were given diets in which parts of additional Leu were replaced with L‐[1–13C]Leu. Breath CO2, plasma Leu and plasma . α‐ketoisocaproate (KIC) after 6h feeding were analyzed using IRMS, AA analyzer and GCMS, respectively. Rates of Leu oxidation (decarboxylation of KIC) were calculated from the rates of 13CO2 production and 13C‐enrichments of plasma KIC. Plasma Leu concentrations and rates of Leu oxidation increased depending on the dosage of additional Leu. However, higher Leu load (20–30%) did not increase the oxidation rates further. Correlation analysis of these two parameters resulted in two linear regression segments that met at a breakpoint (0.8 mM of plasma Leu), suggesting saturation of its catabolic pathway above the breakpoint. The breakpoint occurred at 5 to 6% of additional Leu, which was less than no observed adverse effect level (NOAEL) of Leu in growing rats. Thus, determination of metabolic capacity would be one of keys to estimates safe intake level of Leu.
Citrulline modulates muscle protein synthesis via S6R and P70S6kinase1 activation in old malnourished rats
Authors: Moinard Christophe , Le Plenier Servane , Perret Christine , Cynober Luc 
Published: June 2021

Introduction:It has been observed that citrulline (CIT) is an activator of muscle protein synthesis in aged malnourished rats. However, the underlying mechanism is not known. The rate of protein synthesis is regulated in part by amino acids (i.e. leucine [LEU]) at the level of the mammalian Target Of Rapamycin (mTOR) pathway. The aim of this work is to evaluate the ability of CIT to modulate mTOR pathway in a model of aged malnourished rats.Material and methods:Thirty aged (19 month‐old) rats were submitted to dietary restriction (50 % of the spontaneous food intake) during 12 weeks. They were then randomized in 3 groups: 10 rats (R group) were killed and 20 other were refed (at 90 % of the spontaneous food intake) for 1 week with a standard diet (R+NEAA) or a CIT‐supplemented diet (5g/kg/d) (R+CIT). The refed groups received isonitrogenous nutrition thanks to addition of non‐essential AA (NEAA) to the control group. An additional group was an absolute control group: healthy old rats fed ad libitum (AL)Animals were sacrificed at postabsorptive state, and the tibialis (T) muscle was removed. P70S6K1 and S6R phosphorylation were determined by Western Blot.Results:Conclusion:For the first time the ability of a non proteic amino acid to activate mTOR pathway is demonstrated. This suggest that CIT could be a major player in the control of nitrogen homeostasis.This work was supported by funds from the Ministry of Research and Technology
Alternate‐day fasting reduces the ratio of visceral to subcutaneous fat by modulating adipose tissue triglyceride metabolism in mice
Authors: Varady Krista A , Roohk DJ , Vissa Madhav , Hellerstein Marc 
Published: June 2021

BACKGROUND: Calorie restriction (CR) regimens reduce visceral adiposity in non‐obese animals. The effects of alternate‐day fasting (ADF) regimens on fat distribution are not known.OBJECTIVE: To investigate the effects of ADF on body fat distribution in mice, and to determine the lipid dynamics underlying redistribution of fat among depots.METHODS: In a 2‐week study, 24 male C57BL/6J mice were randomized to 1 of 2 groups: ADF‐100% (24 h of 100% CR, alternating with 24 h ad libitum feeding) or control (ad libitum fed everyday).RESULTS: Body weight did not differ between groups. The epidydimal (visceral) fat depot of the ADF‐100% group was 31% smaller (P < 0.01), while the inguinal (subcutaneous) fat depot was 45% larger (P < 0.01), compared to controls. Absolute TG synthesis in the ADF‐100% group was higher (P < 0.05) in inguinal fat and lower (P < 0.05) in epidydimal fat than in controls. No differences in net lipolysis were observed between groups in either fat pad. Net TG accumulation in ADF‐100% mice was higher (P < 0.05) in inguinal fat and lower (P < 0.05) in epidydimal fat, relative to controls. Epidydimal fat cells were 56% smaller (P < 0.05) in the ADF‐100% group, post‐treatment, relative to controls.CONCLUSION: These findings suggest that short‐term ADF redistributes adipose TG from visceral to subcutaneous depots by augmenting TG synthesis in inguinal fat, while lowering it in epidydimal fat.
Septic shock: A role for RGS proteins?
Authors: Hendriks‐Balk Mariëlle , Tjon‐Atsoi Moenira , Mathy Marie‐Jeanne , Hajji Najat , Michel Martin , Peters Stephan , Alewijnse Astrid 
Published: June 2021

The profound hypotension in septic shock patients is difficult to treat as they display depressed vascular responses to α‐adrenergic agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. Recently, LPS‐induced cardiac failure was found to be associated with upregulation of the Regulator of G protein Signalling (RGS) proteins RGS4 and RGS16 (Cardiovasc Res 53:156, 2002).In this study we investigate the effects of LPS exposure on vascular contractility in general and the role of RGS proteins in the LPS‐induced vascular alterations.Exposure of rat aortic rings to various LPS concentrations (1, 3, 10, 30 μg ml−1) for 22 hours had differential effects on the contractile responses to agonists at four distinct G‐protein coupled receptors. Phenylephrine‐ and angiotensin II‐induced contraction was reduced whereas serotonin‐induced contraction was enhanced. The endothelin‐1‐induced contraction was unaffected. Concomitantly, LPS treatment increased the RGS16 mRNA expression level both in aortic rings and cultured vascular smooth muscle cells (VSMCs) but RGS2, RGS3, RGS4 and RGS5 was unaffected. Regulation of RGS16 mRNA in VSMCs was also time‐ and concentration‐dependent. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses in an LPS model of septic shock.
Symadex™, a FLT3 kinase inhibitor, is metabolized by aldehyde oxidase
Authors: Goodman Keith , Duncan Kenneth , Locniskar Ann , Ajami Alfred 
Published: June 2021

Symadex™ (C‐1311) is a potent inhibitor of FMS‐like tyrosine kinase 3 (FLT3). This imidazoacridinone class compound has started development for oncology indications and is being investigated as a therapy for autoimmune diseases. A major circulating metabolite is an oxygenated species (+16 amu) that is found in plasma and urine after oral and intravenous administration.Aldehyde oxidase (AO) is a molybdenum‐containing enzyme present in liver that catalyzes the oxidation of a wide variety of nitrogen containing heterocycles. Data are presented from in vitro incubations with microsomes, liver cytosol, inhibitors specific for AO, and an isotopic labelling experiment to confirm the role of AO in the oxidation of Symadex™.The proposed metabolite was synthesized and its structure verified by NMR. It was shown to have similar chromatography, spectroscopy, and MS/MS fragmentation compared to the in vitro and in vivo derived metabolite. The structure was consistent with an oxidation catalyzed by AO.This non‐CYP mediated transformation is important for the metabolism of imidazoacridinones and likely plays a significant role in the metabolism of other tyrosine kinase inhibitors with imidazole and related susceptible heterocyclic scaffolds. This work presents a comprehensive investigation into this novel route of drug metabolism.
Impaired prostaglandin E2 (PGE2) production in urothelial cells from an interstitial cystitis patient
Authors: Rastogi Prerna , Dorokhov Nikolay , Rickard Alice , McHowat Jane 
Published: June 2021

SR22 cells are from the inflamed area of the bladder of a patient with interstitial cystitis (IC), a disease characterized by increased activated mast cells in the bladder wall and compromised urothelial function. Since the urinary content of tryptase is increased in IC, this study determined whether there was impairment of prostaglandin E2 (PGE2) release from SR22 cells in response to tryptase. Responses in SR22 cells were compared to responses observed in PD07i cells derived from a healthy bladder. Interestingly, although PAR‐2 density was significantly lower in SR22 cells, tryptase stimulation of either SR22 or PD07i resulted in a similar increase in phospholipase A2 activity and arachidonic acid release. However, SR22 cells did not show any increase in PGE2 release in response to tryptase, whereas there was a significant increase in PGE2 release from tryptase‐stimulated PD07i. The levels of COX‐1 and COX‐2 mRNA expression were equivalent, but mRNA expression of PGE synthase was considerably lower and of hydroxyprostaglandin dehydrogenase was considerably higher in SR22 compared to PD07i, suggesting that both decreased production and increased metabolism could responsible for the lack of PGE2 response. Since PGE2 is cytoprotective, these data may explain why IC is associated with impaired ability to resist cell damage and warrants further investigation.Supported by DK66119 (JM)
In vivo mitochondrial function varies with muscle and training status in young adults
Authors: Larsen Ryan Godsk , Callahan Damien M. , Lanza Ian R. , Kent‐Braun Jane A. 
Published: June 2021

Phosphorus magnetic resonance spectroscopy (MRS) was used to measure the effect of training status on skeletal muscle oxidative capacity (Vmax), an in vivo marker of mitochondrial function, in two morphologically‐distinct human muscles‐tibialis anterior (TA) and vastus lateralis (VL).Methods: Healthy, untrained (7F, 7M, 26±1 yrs, mean±SE) and trained (7M, 1F, 28±1) adults performed a 16‐s and a 24‐s maximal voluntary isometric contraction of the TA and VL, respectively, while intracellular phosphocreatine (PCr) concentration was measured by MRS. The rate constant of post‐contraction PCr recovery (kPCr) was determined and used to calculate Vmax (kPCr · resting [PCr]).Results: There were main effects of muscle and group on Vmax and kPCr such that oxidative capacity was higher in VL than TA (p≤0.046), and higher in trained than untrained (p<0.0001).Conclusion: This study provides novel evidence of muscle‐specific differences in in vivo mitochondrial function that is independent of training status.
Improvement of Ca2+ Transport and Muscle Relaxation in Skeletal Muscle From Sarcolipin Null Mice
Authors: Tupling A. Russell , Hussain Dawar , Trivieri Maria G. , Babu Gopal J , Backx Peter H. , Periasamy Muthu , MacLennan David H. , Gramolini Anthony O. 
Published: June 2021

Sarcolipin (SLN) is an inhibitor of SERCA type pumps by direct binding. To demonstrate whether ablation of SLN in skeletal muscle might impair skeletal muscle function directly, we analyzed skeletal muscle contractility and Ca2+ uptake in SLN null mice (KO). SLN protein expression in wildtype mice (WT) was found to be highest in the quadriceps and gastrocnemius (G) followed by the diaphragm and soleus (Sol), whereas very little SLN expression was seen in the tibialis anterior or extensor digitorum longus (EDL). No SLN protein was detected in muscles from KO. Compared with WT, Ca2+ uptake was increased in the G and Sol of KO muscles, while no significant difference was observed in the EDL. Contractility measurements of WT and KO Sol and EDL muscles were performed. No differences were seen between WT and KO muscles in peak tetanic force; however, peak twitch force was lower in KO by ~28% but only in Sol. Maximum normalized (s‐1) rates of contraction (+dF/dt) and relaxation (−dF/dt) measured during a twitch contraction were significantly higher in Sol of KO compared with WT (+dF/dt, 50.2±5.9 in KO vs 30.9±1.0 in WT; −dF/dt, 7.2±0.5 in KO vs 5.2±0.5 in WT). There were no differences between KO and WT in +dF/dt or −dF/dt in EDL. These results show that SLN regulates muscle contractility in mouse slow twitch fibers by inhibiting SERCA function and reducing Ca2+ uptake.Supported by HSFO (DHM, PHB, AOG), CIHR (DHM, PHB) and NSERC (ART).
Phosphatidylinositol‐4, 5‐bisphosphate has an inhibitory action on native TRPC6 activity in arterial myocytes
Authors: Albert Anthony , Saleh Sohag , Large William 
Published: June 2021

It is generally accepted that TRPC3/C6 and C7 cation channel proteins are activated by diacylglycerol (DAG) via a protein kinase C (PKC) independent pathway but there is no clear evidence on how this DAG‐ mediated process occurs. However a precursor of DAG, phosphatidylinositol‐4, 5‐bisphosphate (PIP2), may also have an important role in modulating the activity of different TRP channels (Voets & Nilius, 2007). We have shown in native rabbit mesenteric arteries that angiotensin II (Ang II) activates a channel, which TRPC6 proteins are likely to represent essential molecular components, through DAG acting via a PKC‐independent mechanism (Saleh et al, 2006) and the present work investigates the effect of PIP2 on this channel activity using whole‐cell and single channel recording.Inclusion of PIP2 in the patch pipette solution and application of PIP2 to the cytosolic surface of inside‐out patches markedly reduced Ang II‐induced whole‐cell cation conductances and channel activity by over 80% at −50 mV. Moreover application of an anti‐PIP2 antibody increased TRPC6 channel activity induced by either Ang II or OAG in inside‐out patches. In cell‐attached patches bath application of the phosphatidylinositol kinase inhibitors wortmannin and LY294002 and the PIP2 scavenger poly‐lysine produced pronounced activation of TRPC6 channel activity.These novel data indicate that PIP2, a substrate of DAG production, has a profound inhibitory action of TRPC6 channel activity in native arterial myocytes indicating that both hydrolysis of PIP2 and production of DAG may be important in activating channels from the TRPC3/C6/C7 subfamily of TRPC proteins.Saleh SN et al (2006). J Physiol 577, 479–495. Voets T & Nilius B (2007). J Physiol 582, 939–944. Work supported by The Wellcome Trust.
Calcium‐dependent and ‐independent mechanisms in the response to oxygen of the chicken ductus arteriosus
Authors: van Cleef Anne NH , Zoer Bea , Cogolludo Angel L , Perez‐Vizcaino Francisco , Villamor Eduardo 
Published: June 2021

We aimed to investigate the relative contribution of voltage‐ and store‐operated Ca2+ channels (VOCC and SOCC respectively), intracellular Ca2+ stores and Ca2+ sensitization in O2‐induced contraction of chicken ductus arteriosus (DA). Isolated DA rings from 15 and 19‐d chicken fetuses (total incubation 21‐d) were mounted in a myograph and exposed to normoxia. In the 19‐d DA, O2‐induced contraction was reduced by the absence of extracellular Ca2+ (28.7% of control contraction) or by the presence of the L‐type VOCC antagonist nifedipine (10 μM; 45.0%); the sarcoplasmic reticulum Ca2+ ATPase inhibitors thapsigargin (0.1 μM; 80.7%) and cyclopiazonic acid (0.1 μM; 22.2%); the SOCC inhibitor SKF 96365 (10 μM; 41.7%); the blocker of Ca2+ release from IP3‐sensitive stores 2‐APB (0.5 μM; 44.0%); and the Rho‐kinase inhibitor hydroxyfasudil (1 μM; 62.3%). The combination of nifedipine, thapsigargin, SKF 96365 and hydroxyfasudil abolished O2‐induced contraction. The L‐type VOCC agonist BAY K8644 contracted the 19‐d DA in a concentration‐dependent manner. This contraction was not affected by O2 tension. The 15‐d DA showed a very weak O2‐induced contraction and did not respond to BAY K8644. In conclusion, our results indicate that Ca2+ entry through VOCC and SOCC, Ca2+ release from intracellular stores and Rho‐kinase‐mediated Ca2+ sensitization contribute to O2‐induced contraction of the chicken DA. Functional immaturity of VOCC appears to play a role in the lack of O2 responsiveness in the immature chicken DA.Supported by "Fundación de Investigación Médica Mutua Madrileña".
Noninvasive imaging of hemorrhage‐induced global ischemia with a transgenic mouse expressing luciferase coupled to hypoxia‐inducible factor (HIF1α)
Authors: Bowman Phillip , Bynum James , Kiang Juliann G. , Baer David , Johnson Joanna , Dubick Michael 
Published: June 2021

Hemorrhagic shock leads to global ischemia, but available blood is distributed unevenly to the body's organs and it is generally accepted that blood is shunted to those organs that maintain critical functions. The organs that become most ischemic and their role in hemorrhagic shock, however, have not been determined. We used the recently described, genetically engineered mouse FVB.129S6‐Gt(ROSA)26Sortm1(HIF1α/luc) Kael/J* (Jackson Laboratories) which has a luciferase gene fused to the region of HIF1α that binds to von Hippel‐Lindau protein in an oxygen‐dependent manner generating a reporter that can be used to monitor oxygen availability in intact tissues. Thus more light would be emitted from ischemic tissue. To determine if this mouse could be used to identify organs affected by hemorrhagic shock, 40 per cent of the calculated blood volume was removed via the submaxillary vein, and the mice were injected with luciferin, anesthetized with isoflurane, and imaged in the Xenogen IVIS 100 Imaging system as a function of time after hemorrhage. The ventral surface of these mice exhibited increased light emitted from the region of the intestine that was most prominent at 3‐6 h after hemorrhage but still evident at 72 h. This technique should allow more detailed studies of those tissues most affected by hemorrhagic shock.*Safran, M.,et. al., Proc Natl Acad Sci U S A 2006;103:105‐110.
Incorporation into the pre‐replication complex activates the Mcm2‐7 replicative DNA helicase for phosphorylation by the S‐phase kinase, Cdc7‐Dbf4
Authors: Bell Stephen P , Francis Laura I , Randell John C. W. , Takara Thomas J 
Published: June 2021

The essential S‐phase kinase, Cdc7‐Dbf4, acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2‐7 replicative helicase. Also known as Dbf4‐dependent kinase (DDK), this kinase is limiting in cells and acts at individual origins of replication at the time of initiation. Here we address the mechanisms that control the specificity of DDK action. We show that DDK preferentially targets a conformationally distinct sub‐population of Mcm2‐7 complexes that are tightly linked to the origin DNA during pre‐replicative complex (pre‐RC) formation. This targeting requires DDK to tightly associate with Mcm2‐7 complexes in a Dbf4‐dependent manner. Importantly, we find that DDK association with and phosphorylation of origin‐linked Mcm2‐7 complexes require prior phosphorylation of the pre‐RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the subset of origin‐bound Mcm2‐7 complexes that will drive replication fork movement during S phase and suggest new mechanisms that could regulate origin activity.This research was supported by funding from the National Institute of General Medical Sciences, the Howard Hughes Medical Institute, and the Leukemia and Lymphoma Society.
Mesenchymal stem cells (MSCs) grown from human prostate cancer can generate atypical glands in vivo
Authors: Croisille Laure , Crisan Mihaela , Yao Veronica , Sun Bin , Parwani Anil , Logar Alison , Matsko Christopher , Bacich Dean 
Published: June 2021

Background / AimsNew therapeutic targets need to be identified in prostate cancer which remains the second cause of cancer related death in US men. Some studies suggest that MSCs may be candidates for cancer transformation in a gastric cancer model. We aim at understanding whether MSCs have a role in prostate tumorigenesis.MethodsCells were obtained and expanded in vitro from 7 CaPs specimens, after fluorescence‐activated cell sorting or after multiple steps of cell adhesion, and eventually characterized by immunocytochemistry and FACS then injected into a xenochimeric mouse model.ResultsMSCs markers (CD9, CD13, CD44, CD73 and CD90) were expressed by 95% to 100% of the cells. Eight weeks after in vivo injection under the renal capsule of immunodeficient mice, in presence of fetal rat UGM, cells from 3 specimens (3/3) generated xenografts, while fetal rat UGM alone and cells alone did not grow. Further analysis of the grafts show the presence of glandular structures with prostate intraepithelial neoplasia features and some glands were of human origin.ConclusionsOur results suggest that MSCs grown from prostate tumors, under appropriate stimulation, are able to generate glandular structures with preneoplastic features. Further characterization of the respective origin of the glands, vessels, and stroma, is mandatory to further elucidate the potential of these MSCs.
The New Classification of Hepatic Segment
Authors: Liu Shuwei , Zhao Zhenmei , Lin Xiangtao , Lou Li , Li Zhenping 
Published: June 2021

ObjectiveTo re‐recognize the intrahepatic vessels and propose a new view of the hepatic segmental classification.MethodsUsing successive sections of 50 adult cadavers and three‐dimensional CT images of hepatic vessel of 300 patients, the branching pattern of intrahepatic vessels and hepatic segments were investigated.ResultsAccording to the branching pattern of intrahepatic vessel, the liver is divided into left and right hemilivers by the median fissure. The right hemiliver is divided into anterosuperior and posteroinferior lobes by the curved right interlobar fissure. The anterosuperior lobe is divided into ventral and dorsal segments by the vertical intersegmental fissure, and the ventral segment is divided into upper and lower subsegments. The posteroinferior lobe is divided into upper and lower segments by the horizontal intersegmental fissure. The left hemiliver is divided into posterosuperior and anteroinferior lobes by the interlobar fissure. The anteroinferior lobe is divided into medial and lateral segments by the intersegmental fissure, and the medial segment is divided into upper and lower subsegments by the horizontal subsegmental fissure. The caudate lobe is separated from other parts of the liver by the arcuate dorsal fissure.ConclusionThis new method of hepatic segment division will be helpful to the precise localization of minimal lesions in the liver.