bluuebunny/crossref_metadata_2025 huggingface dataset:
Purinergic receptors stimulate Calcium transport in pancreatic duct cells
Authors: Novak Ivana , Hansen Mette Rud , Krabbe Simon
Published: June 2021

Normal pancreatic juice contains <1 mM concentrations of Ca2+, but little is known about Ca2+ homeostasis, which may be an important function of duct epithelium. Our aim was to investigate whether duct cells from human and rat pancreas express Ca2+‐transporting and Ca2+‐binding proteins and whether purinergic receptors regulate Ca2+ transport, as is known for anion transport. Expression analysis revealed that pancreatic ducts of rat and human duct cell line CFPAC‐1 (also PANC‐1 and Capan‐1) express the Na+/Ca2+ exchanger (NCX), as well as the Ca2+‐sensing receptor and the Ca2+‐binding protein calbindin 9kD. These proteins localize to the luminal and lateral membranes of pancreatic duct epithelia. Ca2+ imaging experiments showed that activation of purinergic P2X and P2Y receptors stimulated NCX in rat and human duct cells. Application of ATP to CFPAC‐1 monolayers stimulated Ca2+ transport from the luminal to the basolateral side. Taken together, these results show that pancreatic ducts express a number of Ca2+‐handling proteins and we suggest that these together with purinergic receptors may play a role in regulation of pancreatic Ca2+ transport.

Support: PhD scholarship, University of Copenhagen and DNSRC 272‐05‐0420
VASP‐mediated Rac 1 activation contributes to cAMP‐induced microvascular endothelial barrier stabilization
Authors: Schlegel Nicolas , Waschke Jens
Published: June 2021

Vasodilator‐stimulated phosphoprotein (VASP) is required for maintenance of endothelial barrier functions. To test our hypothesis that VASP is involved in regulation of Rac 1 activity, we studied cAMP‐dependent Rac 1 activation. Both, inhibition of Rac 1 activation by NSC‐23677 as well as of protein kinase A (PKA) by PKI completely blunted forskolin/rolipram (F/R)‐mediated cAMP increase to stabilize barrier functions measured as transendothelial resistence (TER). Because this indicates that PKA/Rac 1 activation is important for barrier stabilization, we tested this signalling pathway in VASP (‐/‐) cells. We found that F/R and isoproterenol reduced permeability measured as FITC‐dextran flux across VASP (‐/‐) monolayers, however, not below baseline levels of wild‐type cells (WT). Moreover, cAMP‐mediated Rac 1 activation was reduced to ~50% of WT levels and both PKA inhibition by PKI as well as PKA anchoring via A kinase anchoring peptides (AKAPs) by HT31 almost completely abolished Rac 1 activation in VASP (‐/‐) and WT endothelium. Accordingly, HT31 significantly reduced F/R‐mediated TER increase in WT cells and completely blocked the protective effect of cAMP on endothelial barrier properties. Our data underline the significant role of cAMP‐mediated Rac 1 activation for endothelial barrier stabilization and demonstrate that both AKAP‐mediated PKA anchoring and VASP are required for this process.
Regulation of V‐ATPase, NBCe1‐A and NBCe1‐B in hippocampus
Authors: Martin Henno , Oehlke Oliver , Roussa Eleni
Published: June 2021

The electrogenic sodium‐bicarbonate‐cotransporter (NBCe1) is a key player in regulation of intracellular pH in several cell types. In the CNS however, modes of intracellular pH regulation reveal considerable differences, compared to other tissues, because neuronal activity is accompanied by rapid pH shifts. Here we have investigated expression and regulation of V‐ATPase, and N‐terminal variants of NBCe1 in mouse brain.

V‐ATPase, NBCe1‐A and NBCE1‐B expression was detected in hippocampus, cerebral cortex, olfactory bulb and cerebellum. However, NBCe1 N‐terminal variants revealed distinct cellular distribution in mouse brain and showed differential activity‐dependent regulation in vitro. After induction of extracellular acidosis or alkalosis gene expression and protein expression of V‐ATPase, NBCe1‐A and NBCe1‐B in hippocampal cultures were not changed. However, acute extracellular acidosis resulted to V‐ATPase and NBCe1‐A redistribution from the cytosol towards the plasma membrane.

The results suggest redistribution of acid‐base transporters as main regulation mechanismus during acid‐base disturbances. Moreover, NBCe1 may be involved in neuronal modulation, and pH regulation during neuronal activity.
Ribosome Dynamics, Nascent Chain Elongation and Induced Fit Mechanisms Enabling Antibiotics Selectivity
Authors: Yonath Ada
Published: June 2021

The ribosome's architecture and its dynamic provide the framework for amino acid polymerization and navigate its required motions. Once produced, nascent protein chains progress through an elongated tunnel that can participate in gating, elongation arrest, trafficking and cellular signaling. When emerging from the ribosome the newly born proteins may undergo chaperon‐aided cotranslational folding, associated with conformational rearrangements of the first chaperone to be encountered. Flexible nucleotides are exploited for creating binding pockets for clinically relevant antibiotics by induced fit mechanisms associated with networks of remote interactions capable of allosteric rearrangements.
The Ultrastructural and Mechanical Analysis of the Dentition of Mice Lacking the NBCe1 Na+/HCO3‐ Cotransporter
Authors: Lacruz Rodrigo S , White Shane , Snead Malcolm L. , Paine Michael L , Ira Kurtz
Published: June 2021

Patients with inherited proximal renal tubular acidosis (pRTA) have a systemic metabolic acidosis and may have in addition extra‐renal manifestations (bone growth delays, ocular defects). Amelogenesis imperfecta (AI) is a group of diverse inherited disorders caused by mutations affecting the secreted enamel‐specific proteins but are not usually associated with other diseases. Recent case reports of patients with pRTA who have mutations in the SLC4A4 gene encoding NBCe1 described enamel defects as part of the constellation of extra‐renal findings. We have recently provided the first evidence that NBCe1 is expressed in polarized rodent dental enamel cells (ameloblasts) suggesting that NBCe1 plays an essential role in enamel formation. Mice with a targeted disruption of the Slc4a4 gene and ablation of NBCe1 protein expression are a good model for studying the role of NBCe1 in enamel formation. We present ultrastructural and mechanical data for the dental hard tissues in NBCe1−/− animals. These animals show hypoplastic enamel with severely disorganized prismatic structure similar to that described for AI, which suggests that abnormal pH homeostasis in the developing enamel results in a disruption of enamel crystallite formation creating structurally weak enamel. Normal ameloblast bicarbonate transport/pH regulation is thus required for proper enamel formation.
Functional characterization of blood pressure‐associated common variants in STK39
Authors: Chang Yen Pei Christy , Chen Junji , Wang Ying , Dorff Sarah , Wade James , O'Connell Jeff , Shuldiner Alan
Published: June 2021

We conducted a genome‐wide association study (GWAS) of systolic and diastolic blood pressure (BP) in Amish subjects and identified several SNPs to be robustly associated with BP in a serine/threonine kinase, STK39. Identifying STK39 as a hypertension susceptibility gene is of great biological implication as emerging evidence indicate that STK39 encoded protein, SPAK, interacts with WNK kinases and cation‐chloride transporters in cell‐based assays and participates in an essential sodium‐transport pathway. We demonstrate that in vivo, SPAK is expressed in the distal nephron, where it may interact with these proteins. While none of the associated SNPs from GWAS alter protein structure, we identified several BP‐associated SNPs located in intronic conserved sequence elements (CEs). When tested in reporter gene assays, one of these CEs demonstrated allele‐specific in vitro transcription activity. Oligonucleotides from this CE also showed allele‐specific binding to nuclear protein(s) in electrophoretic mobility shift assays. Hence STK39 may influence BP by allele‐specific DNA‐protein interaction that modifies SPAK expression and renal sodium excretion.
Mineralcorticoid Receptor (MR) Antagonism Attenuates Glomerular Filtration Barrier Remodeling in the Transgenic Ren2 Rat
Authors: Whaley‐Connell Adam , Habibi Javad , Wei Yongzhong , Gutweiler Alex , Jellison Jessica , Wiedmeyer Charles E , Ferrario Carlos M , Sowers James R
Published: June 2021

Renin‐angiotensin‐aldosterone system (RAAS) activation is associated with glomerular filtration barrier/podocyte injury & accompanying proteinuria. To explore potential MR mediated renal effects, Ren2 transgenic rats, which manifest increased tissue RAAS activity with elevated serum aldosterone levels, were used. Six week old Ren2 and Sprague‐Dawley rats were treated with a low dose of the MR antagonist spironolactone (SP)(0.24 mg/day) or vehicle for 21 days. Ren2 rats exhibited albuminuria, loss of podocyte‐specific proteins and podocyte foot‐process effacement on ultrastructural analysis, along with reduced expression of mediators of insulin metabolic signaling. Ren2 kidneys also manifested increased NADPH oxidase/ROS and redox‐sensitive Rho kinase activation in conjunction with enhanced renal tissue levels of MR, angiotensin type 1 receptor, angiotensin II and angiotensin‐(1‐12). SP treatment reduced albuminuria and tissue RAAS expression and improved podocyte structural and protein integrity along with improvements in insulin metabolic signaling. MR blockade directly provides protection in this model of renal RAAS activation, in part, through reductions in redox‐sensitive ROK impairment of insulin metabolic signaling and glomerular remodeling.

Supported by NIH R01 HL73101‐01A1 (JRS) & HL‐51952 (CMF), VA Merit (JRS), VA VISN 15 (AWC), & Missouri Kidney Program (AWC).
Microvascular Loss and Progression of Renal Injury in Renovascular Disease: Renoprotective Effects of VEGF
Authors: Chade Alejandro R. , Iliescu Radu
Published: June 2021

Chronic renal artery stenosis (RAS) induces renal functional impairment by promoting microvascular (MV) endothelial dysfunction, damage, and loss, which contributes to the progression of renal injury. Vascular endothelial growth factor (VEGF), crucial for preservation of microvasculature, promotes endothelial repair and modulates renal fibrosis.

We hypothesized that administration of VEGF would preserve the renal MV architecture and function during the evolution of RAS.

RAS was induced in 12 pigs. VEGF‐165 was intra‐renally infused at the onset of the stenosis (RAS+VEGF, 0.05 ug/kg) in 6 pigs monitored for 6 weeks, and compared to normal controls (n=6). Additional RAS+VEGF pigs underwent revascularization (RV) after 6 weeks, VEGF repeated (RAS+VEGF+RV), and observed for 4 more weeks (n=3). Normal and RAS+RV animals (n=3) served as time‐controls.

Single‐kidney hemodynamics and function were assessed in vivo using ultra‐fast CT after 6 and 10 weeks. Renal MV architecture was investigated ex‐vivo using 3D micro‐CT and CD31 immunostaining, and morphology evaluated in cross‐sections stained with trichrome.

The degree of RAS and increase in blood pressure were similar in RAS and RAS+VEGF animals. However, decreased RBF, perfusion, and GFR in RAS were normalized in RAS+VEGF and accompanied by augmented renal capillary density and decreased fibrosis. Furthermore, RV normalized GFR and blood pressure only in RAS+VEGF+RV pigs.

A single intra‐renal infusion of VEGF improved renal function and decreased fibrosis of the stenotic kidney. This study shows the feasibility of a novel targeted therapeutic approach and supports renoprotective effects of VEGF during the evolution of RAS by preserving renal function not only at the early stage of the disease, but also by improving the responses to revascularization.
Mechanism of CCK‐induced oxytocin release from hypothalamic synaptosomal preparation
Authors: Altimari Michael J. , Patel Niraj , Ponnam Bala S. , Haldar Jaya
Published: June 2021

Previously we have reported that both KCl and CCK‐8 cause oxytocin release from spinal cord synaptosomes. We have also demonstrated the involvement of mitochondria in this release process. Mitochondria could modulate OT release either as a source of Ca2+ or as a source of ATP or both. The current experiments were designed to determine if ATP has some modulatory role in this release mechanism. Present experiments were conducted with hypothalamic synaptosomes prepared from hypothalamic block obtainedfrom freshly sacrificed rats containing both PVN and SON. Synaptosomes were purified by discontinuous percoll gradient (23%,15%,10% &3%). The interface between 23% &15 % was collected and used for the release study. For the current experiments ATP was used as a stimulant. ATP in a concentration of 10nM, 50 nM and 100 nM caused release of oxytocin 90%,364% & 77% rtespectively.. Thus, these results suggest that inhibition of oxytocin release from spinal cord synaptosomes following treatment with electron transport chain inhibitors was probably due to lack of ATP and not due to lack of Ca2+
Differential fetal exposure to inflammatory assaults in Wistar and Zucker rats
Authors: Palmgren Muriel S. , Corll Conni , Svec Frank , Porter Johnny R.
Published: June 2021

Periodontal disease has been linked to cardiovascular disease and preterm birth in humans. Oral inflammation is thought to accelerate inflammation at other sites. An animal model is needed to test this hypothesis. We propose that the intensity of inflammatory response in different strains of rats affects the fetus differently. If so, such differences might explain differences seen in studies of humans. We injected pregnant Wistar (W) and Zucker (Z) rats with 1 mg/kg of lipopolysaccharide (LPS) or saline on gestation day 18. Serum and amniotic fluid (AF) were collected 4 hours later. Saline‐injected rats had no response. Zucker rats had higher TNFα (serum=71,977 pg/ml; AF=161pg/ml) and IL‐6 (serum>200,000 pg/ml; AF=1,985 pg/ml) than Wistar rats (TNFα: Serum=599 pg/ml; AF<15 pg/ml; IL‐6: Serum=22,778 pg/ml; AF<15 pg/ml). Zucker rats died or were moribund by 4 hours. Gestation was not shortened in LPS‐treated Wistar rats. The Zucker fetus was exposed to cytokines at 4 hours post‐injection while the Wistar fetus was not. The Zucker rat is a better model for inflammatory assaults on the fetus. Supported in part by Research Grant No. 21‐FY1251 from the March of Dimes Birth Defects Foundation.
The ontogeny of dactylin expression in the embryonic mouse
Authors: Maze TD , Bradley Amber , Maze Jennifer
Published: June 2021

The current study is designed to characterize the ontogeny of dactylin expression in the developing mouse embryo. Dactylin is an important gene for proper digit formation. It is a member of the F‐box/WD40 gene family and is involved in the ubiquitin ligase process that targets specific proteins for degradation. Mouse dactylaplasia has similar phenotypic characteristics to humans with split hand/ spilt foot malformation, furthermore dactylin, which has been mapped to chromosome 19 in mice, is syntenic to the SHFM3 region in humans on chromsome 10q24. Understanding the ontogeny of dactylin is important because it allows us to look at specific time periods critical for the development of digits. Mouse embryos were collected prior to digit formation (E8 and E10), during digit formation (E12), and after digit formation is complete (E14) to determine at what point dactylin expression was turned on and off. Once the embryos were collected the limb buds or entire limbs were removed, total mRNA isolated, and RT‐PCR was used to determine gene expression. We found dactylin expression to be present at all time periods examined. In order to get a more complete ontogeny, we are currently expanding the original time frame to E2 through E21. This work was supported by a Lander University Foundation Grant.
Altered expression of paternally imprinted genes is associated with fetoplacental demise in the BPH/5 model of pre‐eclampsia (PE)
Authors: Woods Ashley Kathryn , Zhou Yi , Sharma Ram V , Davisson Robin L
Published: June 2021

Pregnancies in BPH/5, a mouse model of PE, are characterized by abnormal placentation and fetal demise starting early in gestation. Paternally imprinted genes, often found in the placenta, have been implicated as regulators of placental and fetal growth. We hypothesized that paternally imprinted genes would be dysregulated in BPH/5. Gene expression microarray studies were carried out on BPH/5 fetoplacental units sonographically scored and categorized into three increasingly severe phenotypic classes based on qualitative and quantitative parameters. The three classes showed a total of 697 dysregulated genes. Three paternally imprinted genes were dysregulated in BPH/5 and were verified with real time PCR. These include: insulin growth factor 2 (Igf2) which was decreased in Class II and III fetoplacental units(0.32 ± 0.13 and 0.26±0.17 fold vs C57, n=3 p<0.05), delta‐like 1 (Dlk‐1) which was decreased in Class II and III (0.08 ± 0.04 and 0.19 ± 0.13 fold vs C57, n=3 p<0.05), and paternally expressed gene 3 (Peg3) which was decreased in Class II (0.22 ± 0.09 vs C57, n=3 p<0.05). Recent studies using knockout mice show that these paternally imprinted genes regulate placental size and depth, suggesting that these genes may be linked to fetoplacental abnormalities in our model of PE.
Effect of L‐NAME on hemodynamic parameters and thoracic aorta tension in rats of severe sepsis
Authors: Xia Man‐li , Huang Yue‐yan , Qian Ling‐bo , Xia Qiang
Published: June 2021

The objective of the present study was to explore the effect of Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME) on hemodynamic parameters and thoracic aorta tension in rats of severe sepsis. We used cecal ligation and puncture (CLP) method to establish severe septic rats, and L‐NAME (5 mg/kg body weight, i.p.), the nitric oxide synthase inhibitor, was injected after CLP for 4 h. In the phase of severe sepsis (20 h after CLP), the carotid artery was cannulated and connected to a pressure transducer to determine mean arterial blood pressure (MABP); Ventricular dynamic parameters were determined following intraventricular cannulation via the carotid artery, including heart rate (HR), left ventricular developed pressure (LVDP), and maximal rise/fall velocity of ventricular pressure (±dP/dtmax); Isolated thoracic aortic rings were mounted on the organ bath and the tension of the vessel was recorded. We found that MABP and ventricular‐dynamic parameters, including HR, LVDP and ±dP/dtmax, were decreased in severe septic rats, which were markedly recovered by treatment with L‐NAME. Vasoconstriction to PE, KCl, and Ca2+ was also markedly increased by L‐NAME in both endothelium‐intact and ‐denuded aorta of severe septic rats. The results indicate that overproduction of nitric oxide (NO) may involve the change of hemodynamic parameters and the vascular hyporeactivity in severe septic rats induced by CLP, and the mechanism of L‐NAME improving hemodynamic parameters and vasoconstriction responsiveness in rats of severe sepsis is mediated by inhibiting the overproduction of NO.
Critical Role of αB Crystalline on Stretch‐Induced FAK Activation in Cardiomyocytes
Authors: Pereira Michelle B. M. , Santos Aline M , Franchini Kleber G.
Published: June 2021

Aim

Focal adhesion kinase (FAK) is activated and plays a role in cardiomyocyte hypertrophy induced by mechanical stress. FAK activation may depend on cooperation with protein partners. We investigated whether interaction with alpha‐B crystalline (αB Crys) regulates FAK activation by mechanical stress in cardiomyocytes.

Methods

αB Crys and FAK expression and phosphorylation at Tyr397 (pFAK) were quantified in neonatal rat ventricular myocytes (NRVMs). FAK/αB Crys interaction was investigated by co‐immunoprecipitation and pulldown assays. αB Crys depletion by RNAi was used to examine its effect on FAK activation.

Results

Cyclic stretch (10%, 2 hs) increased pFAK (2‐fold) and enhanced αB Crys expression (4‐fold) and phosphorylation at Ser59 (3‐fold). Stretch also induced FAK/αB Crys association. Pulldown assays with recombinant FAK domains indicated that the interaction with αB Crys is mediated by FAK C‐terminal domain. FAK immunostaining was coincident with myofilaments in non‐stretched while in stretched cells it was predominantly located in the nuclei. αB Crys was predominantly located at the nuclei of stretched NRVMs, coincident with FAK staining. Depletion of αB Crys markedly reduced FAK activation and re‐location to the nucleus.

Conclusion

This study demonstrates that αB Crys associates and regulates FAK activation in cardiomyocytes in response to mechanical stress.
Effects of cell‐free layer width and its variability on wall shear stress in arterioles
Authors: Namgung BumSeok , Ong Peng Kai , Johnson Paul C , Kim Sangho
Published: June 2021

It has been reported that the cell‐free layer width and its interface roughness with erythrocyte core may significantly influence wall shear stress in microcirculatory vessels. However, to date, the quantitative information on how the cell‐free layer width and/or its variability affect the wall shear stress is limited. In this study, temporal variation data of the cell‐free layer width in arterioles (ID 50‐29 μm) in rat cremaster muscles were used to investigate effects of the layer width and its variability on the wall shear stress. The coefficient of variation (CV) of the cell‐free layer width was used to quantify the interfacial variability between the cell‐free layer and erythrocyte core. Velocity gradient in the cell‐free layer was assumed to be linear, and thus the wall shear stress was determined with plasma viscosity and slope of the linear velocity gradient. This study was performed under normal (285.7 ± 37.9 s−1) and reduced (14.4 ± 0.5 s−1) flow conditions with and without erythrocyte aggregation induced by dextran infusion. The level of rat erythrocyte aggregation was adjusted to levels found in normal human blood. As expected, the arteriolar wall shear stress decreased exponentially with mean cell‐free layer width. There was a significant effect (P < 0.05) of erythrocyte aggregation on the variability (CV) of the cell‐free layer width, resulting in a significant increase (P < 0.05) of the wall shear stress.
Optimizing the calculation of alveolar capillary conductance and pulmonary capillary blood volume with carbon monoxide and nitric oxide in humans
Authors: Ceridon Maile Lani , Bilezikian Jordan A , Olson Thomas P , Beck Kenneth C , Johnson Bruce D
Published: June 2021

The classic method for determining alveolar‐capillary membrane conductance (DM) and pulmonary capillary blood volume (VC) involves measurement of lung diffusing capacity for carbon monoxide (DLCO) at multiple inspired O2 tensions (FIO2). Recently, the technique has been simplified by simultaneously measuring DLNO (nitric oxide) to determine values at a single FIO2. Controversy exists over equations for calculation of θCO (reaction rate of CO with Hgb) and the correct diffusion ratio for NO and CO (α) in the calculation of DMCO. The purpose of this study was to investigate how various published equations and ratios influence DM & VC using both the multiple and single FIO2 methods. Eight healthy males were studied. DLCO & DLNO were measured using the rebreathe method (inspired vol. ~75% of inspiratory capacity, FIO2 18, 35, & 55%). DM & VC were first calculated using five reported equations for θCO. Once DM & VC were optimized based on reported predicted values, the DLNO method was compared using a range of α values. Using the multiple FIO2 method, depending on the equation for θCO, DM ranged from 47 to 103 ml/min/mmHg, and VC from 74 to 102 ml. For the calculation of DM & VC using the DLNO method, α ranged from 1.5 to 4.0 in order to optimize DM & VC values to the classic method. The determination of DM & VC is critically dependent on the equations used for θCO which in turn affects the optimal ratio used to correct the DLNO method. NIH HL71478
Case report: Anatomic variant of forearm arteries and its clinical correlation
Authors: Kumka Myroslava
Published: June 2021

Methods

Traditional macro‐micro dissections of 30 upper limbs from 15 embalmed cadavers of both sexes and of different ages were performed.

Summary of results: An atypical course and branching pattern of forearm vessels was found unilaterally in one male cadaver. The ulnar artery originated as one of the two terminal branches of the brachial artery and was significantly smaller than commonly seen. It descended superficially, covered only by the skin and the anterbrachial fascia. The branching pattern of the radial artery was also atypical. A common interosseous artery which typically arises from the ulnar artery, in this case emerged as a short branch of the radial artery distal to the radial tuberosity. While passing toward the interosseous membrane, the common interosseous artery was separated from the median and anterior interosseous nerves by the ulnar head of the pronator teres muscle and branched out into the anterior and posterior interosseous and ulnar recurrent arteries that supply the major part of the anterior forearm region.

Conclusion

This anatomical variant of forearm arteries should be considered in diagnosis and treatment since it could complicate radiographic, surgical, and orthopaedic procedures in the forearm, such as radial artery screening for grafting purposes, ligation of a lacerated radial or ulnar artery, and assessment of forearm vessels by color Doppler ultrasonography.
Presence of Markers of Femoroacetabular Impingement in the Asymptomatic Population
Authors: Raynor Charys Megan , Bryant Dianne , Spouge Alison , Birmingham Trevor B , Willits Kevin
Published: June 2021

Purpose

To determine: 1) the prevalence of femoroacetabular impingement (FAI) in asymptomatic individuals, and 2) whether there is an association between the presence of FAI and age and degenerative disease. Methods: 88 volunteers were verified as asymptomatic through physical examination and then underwent an MRI of the hip. Different criteria values were used to classify the presence or absence of cam (alpha angle of 45, 50 and 55 degrees) and pincer (centre‐edge (CE) angle of 35 and 40 degrees) impingement.

Results

The prevalence of FAI ranged from 23.9 to 67.0% depending on the classification criteria applied. There was no significant association between FAI and age. Prevalence of FAI, identified using high criteria values (55 degree alpha angle and 40 degree CE angle), predicts the presence of acetabular cartilage lesions. Prevalence of FAI, identified using lower criteria values, was not a significant predictor of degenerative disease.

Conclusion

FAI is prevalent in the asymptomatic population. Further research is necessary to verify the validity and reliability of criteria used to diagnose impingement and to determine the validity of the diagnosis. Support: AANA and SIRF.

Grant Funding Source

Internal
Accuracy and precision comparison of the hemocytometer and automated cell counting methods
Authors: Stone Laurel Rebecca , Gray Diane R , Remple Krissy , Beaudet Matthew P
Published: June 2021

The hemocytometer is the most widely used device for determining cell concentrations, requiring consistent criteria and tenacity to obtain measurements correctly and reproducibly. As an alternative, the Countess(tm) Automated Cell Counter employs digital imaging and an image analysis algorithm to identify and enumerate cells in a sample. To compare the performance of these two methods, the accuracy, precision, and effective ranges were determined for total cell count and percent viability. First, using a standardized bead solution, the accuracy and precision of glass and disposable hemocytometers were measured and found to be 30% and 5% different from the anticipated value, respectively. Subsequently, disposable hemocytometers and the automated counter were used to determine total cell numbers and percent viability for multiple cell types over a range of cell concentrations. Using a Student's t‐test, the results showed the Countess(tm) instrument measured cell concentrations and viability as accurately and precisely as the disposable hemocytometer. Additionally, the effective concentration range for the Countess(tm) instrument was two times greater than the hemocytometer, and the viability range matched the hemocytometer. The Countess(tm) Automated Cell Counter overall produces results much more rapidly without the problem of operator tedium and fatigue or compromised accuracy and precision.
Is Increased IL‐6 the Result or Cause of Smell Loss in Patients with Hyposmia?
Authors: Henkin Robert I. , Schmidt Loren , Velicu Irina
Published: June 2021

IL‐6 is a neuroactive cytokine which is elevated in plasma of patients with many inflammatory conditions; it is considered either a result of various neurological disorders or a non‐specific change associated with various diseases or with stress itself. We compared IL‐6 in plasma, urine, saliva and nasal mucus in 59 patients with hyposmia related to diseases of a chronic nature including post viral infection (PIHH), post head injury (HI), allergic rhinitis (AR) and burning mouth syndrome (BMS). Nasal inflammation is present only in PIHH and AR. IL‐6 was elevated in plasma in all hyposmic patients but elevated in saliva and nasal mucus only in patients with PIHH, HI and BMS. Elevated IL‐6 could cause smell loss through its action on NF ‐ ?B, ATP ‐ ubiquitin‐dependent proteolytic pathways, TNF‐á or other cytokine pathways which activate cellular apoptosis. Neuropoietin, an IL‐6 related cytokine, affects signaling through ciliary neurotrophic factor receptor and could inhibit smell function since inhibition of several ciliary factors have induced smell loss. These relationships suggest that IL‐6 may be either the result of or a direct cause of hyposmia. These studies support the concept that hyposmia has a biochemical basis and that discovery of the role IL‐6 plays in hyposmia offers an opportunity to learn about its underlying biochemical pathology and to establish methods to treat diseases which cause it.
Enzymology of DNA Repair in the radiation‐resistant extremophile Deinococcus radiodurans
Authors: Cao Zheng , Shadrick William R , Julin Douglas
Published: June 2021

The bacterium Deinococcus radiodurans exhibits extraordinary resistance to the effects of ionizing radiation, and other genotoxic agents. We are using genetic and biochemical approaches to define the enzymatic pathways by which D. radiodurans repairs DNA damage.

D. radiodurans genome loci DR1902 (RecD) and DR1572 (the putative helD gene) encode DNA helicases. Cells with a helD deletion are more sensitive than wild‐type to hydrogen peroxide and methyl‐methanesulfonate, but they are not sensitive to gamma or UV irradiation. Both helicases have a protein domain that is homologous to other superfamily 1 helicases, and an additional N‐terminal domain. The N‐terminal domain of HelD is essential for DNA unwinding by this enzyme. We are attempting to purify the helicase domain of RecD as defined by limited proteolysis, and to examine the kinetics of DNA unwinding by the domain and the full‐length RecD protein. Genome locus DR1126 encodes RecJ, a single‐strand‐specific exonuclease in the RecF pathway for DNA recombination and repair in E. coli. We have deleted the gene from the D. radiodurans genome by standard homologous recombination techniques. We are unable to delete the gene from each of the ~10 chromosomal copies in the cell, suggesting that recJ may be an essential gene. Cells carrying the partial deletion are more sensitive to UV irradiation than wild‐type, suggesting an important role for RecJ in D. radiodurans.
Mutational analysis of a putative base flipping residue R275 in human thymine DNA glycosylase
Authors: Maiti Atanu , Drohat Alexander C.
Published: June 2021

Thymine DNA glycosylase (TDG) is a base excision repair enzyme that excises T from G·T mispairs and removes other lesions, exhibiting specificity for damage at CpG sites. DNA glycosylases use nucleotide flipping to find lesions and cleave the base‐sugar bond. Typically, a bulky side chain penetrates the DNA, filling the space vacated by the flipped nucleotide. Our crystal structure of human TDG catalytic domain bound to abasic DNA shows that an arginine (R275) plugs the helical gap and contacts two phosphates flanking the abasic sugar (Maiti A, et al., Proc Natl Acad Sci 105: 8890‐8895). Here, we examine the role of R275 in the catalytic mechanism of TDG. Isothermal titration calorimetry (ITC) experiments show that substrate (analog) binding affinity is significantly lower for R275A‐hTDG versus hTDG, and lower still for R275L‐hTDG. Single turnover kinetics experiments show that kmax is significantly lower for R275A‐hTDG versus hTDG, and lower still for R275L‐hTDG. Our results are surprising, because two related enzymes, mismatch‐specific uracil glycosylase and uracil DNA glycosylase have leucine at the position corresponding to R275 of hTDG. We find that mutation of R275 has no effect on steady‐state turnover, kcat, suggesting that R275 cannot account for the exceedingly slow product release observed for hTDG. This work was supported by an NIH grant (to ACD).
An in vitro assay for double strand break repair
Authors: Klugman Sarit A. , Norais Cedric , Hamilton Lindsay L. , Cox Michael M.
Published: June 2021

We have developed an automated high‐throughput screen for conditions and extracts that promote double strand break repair in vitro. The screen is based on the facile transformation and metabolic properties of Acinetobacter baylii. The extracts are derived from the very radiation‐resistant bacterium Deinococcus radiodurans, as well as from some E. coli strains that have evolved resistance to irradiation. Results of the screen will be presented.
Characterization of metal‐sensing riboswitches
Authors: Dann Charles E , Wakeman Catherine A , Winkler Wade C
Published: June 2021

Many cis‐acting small regulatory RNAs that bind metabolites, termed riboswitches, have been identified in recent years. The binding of a metabolite to riboswitches results directly in genetic regulation of its transcript through diverse mechanisms affecting transcription termination, translation initiation, mRNA stability and splicing. We have focused on the M‐box aptamer domain, previously referred to as the ykoK orphan riboswitch. Through a convergence of many lines of biochemical, biophysical and structural data, we have demonstrated that this new class of riboswitch senses divalent cations. The mechanism of metal ion sensing based on our 2.6 Åcrystallographic structure will be presented as well as supporting data for metal binding at key sites in the RNA. This work represents a substantial step forward in our thinking about potential ligands sensed by riboswitches and raises the possibility that additional metal‐sensing RNAs could exist. Funding provided by the Searle Scholars Program, the UT Southwestern Medical Center Endowed Scholars Program, the Welch Foundation, the Sara and Frank McKnight Fund for Biochemical Research, Indiana University CoAS, and NIH Grant # 5R01GM081882‐02 (WCW).
A reversed approach for finding small RNAs regulating genes of interest
Authors: Mandin Pierre , Gottesman Susan
Published: June 2021

In E. coli, the largest class of small regulatory RNAs (sRNAs) binds to the RNA chaperone Hfq and regulates the stability and/or translation of specific target mRNAs. We developed a new method that allows screening for sRNAs regulating genes of interest, using translational fusions of the 5'extremities of those genes to the lacZ reporter gene. A multicopy plasmid library of the E. coli genome can then be screened to determine if over‐expression of certain sRNAs affects the activity of the fusion. This screening method was first applied to the transcriptional regulators DpiB/A. RybC, a small RNA of previously unknown function was found to negatively regulate the expression of dpiB. Using mutants in the dpiB‐lacZ fusion and compensatory mutations in the RybC sRNA, we demonstrated that RybC acts post‐transcriptionally and directly on the dpiBA mRNA. This approach has now been generalized to probe the post‐transcriptional regulation of sigma factors by small RNAs; initial results suggest a complex network of regulation, supporting the usefulness of this approach and the multiple layers of regulation present in bacteria.
Involvement of c‐Cbl a ring finger E3 ubiquitin ligase on cyclic GMP dependent protein kinase ubiquitination and degradation
Authors: DEY NUPUR B , LINCOLN THOMAS M
Published: June 2021

Cyclic GMP‐dependent protein kinase‐1 (PKG‐1) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell‐specific gene expression. PKG‐I expression in cultured smooth muscle cells (SMC) depends on culture conditions and is inhibited by inflammatory cytokines such as interleukin‐I and tumor necrosis factor‐α, which are known to stimulate Type II NO synthase (iNOS) and subsequent elevation of cGMP. Chronic elevation of cGMP, as seen in inflammatory conditions, triggers ubiquitination and proteasomal degradation of PKG‐Iα in smooth muscle. Previously we reported that the suppression of PKG‐I protein expression in SMC is triggered by the ubiquitin/26S proteasome pathway. In the current study we found that cGMP elevation down‐regulates PKG‐Iα, but not the I ?, isoform. PKG catalytic activity (autophosphorylation) was required for the suppression of protein expression. Mutation of the known autophosphorylation sites of PKG‐Iα to alanine uncovered a specific role for autophosphorylation of serine‐64 in cGMP‐dependent ubiquitination and suppression of PKG protein expression. We further explored the involvement of specific E3 ubiquitin ligases for this process. We examined the relationship between cCbl and PKG‐1α and the involvement of the ring finger E3 ligase Cbl in the process of ubiquitination. We observed that as a potential PKG‐1α ubiquitin E3 ligase, Cbl forms a complex with and ubiquitinates PKG. These results suggest that down regulation of PKG 1α is triggered by serine‐64 autophosphorylation and then subsequent binding of the ubiquitin E3 ligase, Cbl.
Experimental model of the interplay between TBL1‐mediated activation and Siah‐1‐induced poly‐ubiquitination of β‐catenin
Authors: Dimitrova Yoana N. , Chazin Walter J.
Published: June 2021

β‐catenin is a critical factor in the Wnt signaling pathway and its function is maintained by balancing activation and degradation signals. TBL1 has been identified as a transcriptional co‐activator of β‐catenin. Remarkably, TBL1 has also been associated with an SCFTBL1‐like E3 ubiquitin ligase, initiated by Siah‐1 upon DNA damage, while Siah‐1 itself functions as a potent E3 ligase. Our studies focus on understanding the complex interplay between TBL1‐mediated activation and Siah‐1‐induced poly‐ubiquitination of β‐catenin.

Protocols for recombinant production of the full‐length TBL1 and Siah‐1 have been developed and the proteins were used in in vitro ubiquitination assays to probe the poly‐ubiquitination of β‐catenin. Our results have shown that Siah‐1 is able to efficiently poly‐ubiquitinate β‐catenin as a simple E3 ligase. Interestingly, the addition of TBL1 is seen to inhibit β‐catenin ubiquitination. In order to understand how TBL1 and Siah‐1 compete for β‐catenin, we have mapped the binding domains in Siah‐1‐β ‐catenin and TBL1‐β‐catenin complexes and characterized the strength of their interactions. Combining our biochemical and biophysical data with computational approaches, we have designed a model of the molecular events regulating the functional state ofβ‐catenin. Support: NIH 5R01GM075156‐04.
Hydrophobic core mutations associated with cataract formation destabilize human gammaD‐crystallin
Authors: Drahos Kate Lauren , King Jonathan
Published: June 2021

The human eye lens is composed of fiber cells packed with crystallins up to 400 mg/ml. Human γD‐crystallin (HγD‐Crys) is a monomeric, two‐domain protein found in the lens nucleus. Previous genetic screens for mutations resulting in cataract in mice identified mutations affecting γ‐crystallins. Three such amino acid substitutions were introduced into HγD‐Crys and the mutant proteins L5S, V75D and I90F were expressed and purified from E. coli. All mutant proteins had decreased thermal stability compared with wild type (WT). Equilibrium unfolding/refolding experiments measured the thermodynamic stability of the mutant and WT proteins. WT HγD‐Crys exhibits a three‐state unfolding pathway with the N‐terminal domain (N‐td) unfolding first, followed by the C‐terminal domain (C‐td). L5S and V75D, both N‐td mutants, displayed three‐state unfolding transitions with populated partially‐folded intermediates. In both cases, the first transition was shifted to lower denaturant concentrations. I90F, a C‐td mutant, exhibited a two‐state unfolding transition. In kinetic unfolding experiments the N‐td's of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded through a two‐state mechanism. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.
Pathogenic P0 mutations are blocked at various quality control check points.
Authors: Tinelli Elisa , Modica Graziana , Domenica Vizzuso , Feltri Maria Laura , Wrabetz Lawrence
Published: June 2021

In peripheral nerve, Schwann cells enwrap axons and form myelin. P0 is the most abundant transmembrane glycoprotein produced by Schwann cells, where it is normally delivered to the myelin sheath. Alterations of the dosage or the sequence of P0 cause diverse peripheral neuropathies, like Charcot Marie Tooth (CMT) or Dejerine Sottas Syndrome (DSS). Deletion of serine 63 (S63del), the first amino acid (aa) of the β‐strand C of the extracellular domain, causes the retention of the protein in the endoplasmic reticulum (ER)‐Golgi complex. Protein quality control in the ER ensures that only correctly synthesized proteins are delivered to the final destination. Here we correlate mutations in aa important for the maturation of P0, with their neuropathy phenotypes. Alteration of the glycosylation site does not alter the trafficking, whereas mutation of aa important for the formation of the disulfide bond cause retention of the mutant proteins in the ER‐Golgi compartment. Interestingly, the retention of mutants in the β‐strand C or in the cysteines of the disulfide bond is mediated by two different sets of chaperones, suggesting multiple check points mediate P0 maturation. Therefore, some P0 mutants exit the quality control and are pathogenic in the myelin sheath, whereas others are retained at various points of maturation in the ER. However, the location of intracellular P0 retention does not define the neuropathy phenotype.
Dual function of plasmin(ogen) system in PrPSc propagation in vitro
Authors: Mays Charles E. , Kim Younghwan , Titlow William , Telling Glenn , Ryou Chongsuk
Published: June 2021

Prions are infectious particles that consist of the misfolded, disease‐associated prion protein, PrPSc, serving as a template to convert the conformation of the cellular prion protein, PrPC. Many lines of evidence suggest that a co‐factor is involved in the conversion process. Recently, kringle domains of plasminogen were identified to interact with PrP. However, the in vivo importance of this interaction in prion pathogenesis remains inconclusive: One study showed a modest prolongation, but another demonstrated a significant decrease of incubation period in plasminogen null mice infected by prions. To investigate the role of plasmin(ogen) system in PrPSc propagation, we studied the changes of PrPSc levels using in vitro systems with or without the addition of plasmin(ogen). Here, we demonstrate that plasminogen increased, but plasmin abolished, PrPSc propagation during protein misfolding cyclic amplification as well as in a prion‐infected cell culture model. The assays inhibiting the interaction of plasminogen with PrP and enzymatic activity of plasmin reversed the induced effects. Although mechanistic details of plasminogen‐induced PrPSc formation are not clear yet, plasmin abolishes PrPSc propagation by cleaving PrPC, which reduces the level of PrPC available for conversion. Our results provide evidence that the dual function of the plasmin(ogen) system regulates PrPSc propagation.
Cytoglobin regulates cell respiration and nitrosative stress through NO dioxygenation and co‐localizes with inducible nitric oxide synthase during vascular injury.
Authors: Halligan Katharine E. , Jourd'heuil Frances , Jourd'heuil David
Published: June 2021

Disposition of the second messenger nitric oxide (NO) in mammalian tissues occurs through multiple pathways including reaction with erythrocyte hemoglobin, red muscle myoglobin, and metabolism by NO dioxygenase activities in non‐striated tissues. Although the newly discovered globin cytoglobin binds molecular oxygen and dioxygenates NO in vitro, the lack of an associated reductase activity has raised doubts about the ability of cytoglobin to dioxygenate NO in vivo. We hypothesize that non‐striated cells can dioxygenate NO and that cytoglobin contributes to this activity. For this purpose, we stably expressed short hairpin RNA targeting cytoglobin in mouse fibroblasts. This resulted in an 80 percent reduction in cytoglobin protein expression. Wild type cells demonstrated intracellular nitrate generation upon addition of exogenous NO that was oxygen‐dependent and cyanide‐inhibitable. The sustained dioxygenation of NO observed over a 30 min exposure to NO was diminished in cells with low cytoglobin expression. Protein nitrosation and heme‐nitrosylation were increased in the absence of cytoglobin. These cells were also more sensitive to NO‐induced inhibition of cell respiration. Normal response to NO could be re‐established through expression of human cytoglobin in the knock down cells. In summary, our study reveals a pivotal role for cytoglobin in cell‐mediated NO dioxygenation to regulate nitrosative stress and cell respiration during conditions of chronic NO exposure. This may impact regulation of NO bioavailability in the vasculature.
Two human holocarboxylase synthetase proteins display similar kinetic properties
Authors: Ingaramo Maria , Beckett Dorothy
Published: June 2021

Human Holocarboxylase Synthetase (HCS) catalyzes transfer of the cofactor biotin to biotin‐dependent carboxylases. Consequently, this enzyme is of fundamental importance for fatty acid synthesis, gluconeogenesis, and amino acid catabolism. In addition, in agreement with its role in transcription regulation, it has been proposed that HCS biotinylates histones. There are two major forms of HCS present in human cells that differ by 58 amino acids at the N‐terminus. In order to determine if these residues have any effect on HCS catalytic activity, both forms were recombinantly expressed, purified, and subjected to biochemical characterization.

The oligomeric state and kinetic behavior of full length HCS and 58‐HCS were characterized. Equilibrium sedimentation experiments indicate that both HCS forms are monomers even in the presence of the intermediate biotinoyl‐5′‐adenylate. The steady state kinetic analysis as a function of biotin, ATP or biotin accepting substrate concentrations indicate that full length HCS and 58HCS behave similarly. Therefore the additional N‐terminal amino acids do not affect catalysis and are not involved in biotin acceptor fragment recognition. Furthermore, the results indicate that the distinct roles of HCS in transcription regulation and metabolism reflect properties other than the fundamental kinetic characteristics of the two enzyme forms.