bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal Medullary Hemodynamics in Response to Contrast Media
Authors: Persson Pontus B. , Seeliger Erdmann , Wronski Thomas , Ladwig Mechthild , Flemming Bert
Published: June 2021

Contrast media induced nephropathy (CIN) is the third leading cause for acute renal failure. Pathophysiology of CIN is poorly understood, but involves hypoxia of the outer medulla. Until today, the role of osmolality of contrast media (CM) is considered the essential feature causing CIN. Here we describe a novel, perhaps most relevant characteristic of CM regarding the pathophysiology of CIN: The rheological properties, i.e. viscosity of CM. In 122 rats, we administered CM of equivalent iodine concentrations but differing physiocochemical properties: the high‐osmolality iopromide was compared to the high‐viscosity iodixanol. In addition, the non‐iodinated substances mannitol (equivalent osmolality to iopromide) and dextran (equivalent viscosity to iodixanol) were also studied. Both types of CM transiently increased renal blood flow. The high‐osmolality agents iopromide and mannitol markedly increased urine production where as iodixanol, which caused less diuresis, significantly enhanced urine viscosity. Only the high‐viscosity agents iodixanoland dextran decreased renal medullary blood flux, erythrocyte concentration, and pO2. Moreover, iodixanol prolonged the tubuloglomerular feedback response and increased plasma creatinine levels to a greater extent than iopromide or dextran. Thus, viscosity of CM may play a significant role in CIN.
Compensatory hyperfiltration and NO in 2k1c and uninephrectomized rats
Authors: Hultström Michael , Helle Frank , Iversen Bjarne M.
Published: June 2021

Afferent arterioles from the non‐clipped kidney from 2k1c rats has an increased nitric oxide (NO) release after angiotensin II stimulation. This study investigated if this could be compensatory hyperfiltration. Rats underwent uninephrectomy (unx), 2k1c‐ or sham‐surgery. Six weeks after the initial surgery the rats were sacrificed. The effect of L‐NAME on renal blood flow (RBF) was assessed with an ultrasound probe. Af were isolated using the agarose infusion method to study the effect of L‐NAME on Ang‐II induced contraction. NO release was studied using DAF‐FM.

RBF was increased in the remnant kidney and in the non‐clipped kidney from 2k1c rats (3.82 ± 0.5 ml/min in controls compared to 8.0 ± 1.61 ml/min in the remnant kidney and 3.2 ± 0.17 and 4.8 ± 0.95 in the clipped and non‐clipped kidney respectively). L‐NAME treatment increased MAP around 30 mmHg in all groups while RBF decreased about 35% in all groups, except in the clipped kidney (−24 ± 3 %). AngII elicited similar contraction in Af from unx and controls. L‐NAME did not alter the contraction. No difference was seen in DAF fluorescence between Af from control and unx following AngII.

In conclusion, our results in vivo indicate NO is of lesser importance in the clipped kidney than in the non‐clipped or in control kidneys. Unx Af does not have an increased AngII elicited NO release.

The study was funded through Helsevest, Norway.
Angiotensin II infusion improves renal function in experimental hyperdynamic sepsis
Authors: May Clive , Wan Li , Langenberg Christoph , Bellomo Rinaldo
Published: June 2021

In a model of hyperdynamic sepsis we found a large increase in renal blood flow (RBF) that was paradoxically accompanied by renal failure, which we hypothesized may be caused by decreased glomerular filtration rate due to efferent arteriolar vasodilatation. We therefore investigated the effect in sepsis of infusion of angiotensin II (Ang II), a preferential efferent arteriolar vasoconstrictor. In 6 conscious ewes, with flow probes around the aorta and renal, mesenteric, coronary and iliac arteries, intravenous live E. Coli. induced hyperdynamic sepsis (hypotension and increased cardiac output). RBF increased (292 ± 61 to 397 ± 74 mL/min P<0.01), but oliguria developed and creatinine clearance decreased (88.7 ± 19.6 to 47.7 ± 21.0 mL/min, P<0.01). Ang II restored arterial pressure and RBF, increased urine output 6 to 8 fold and doubled creatinine clearance. Importantly, Ang II had no adverse affects on vital organ blood flows or biochemical variables. In experimental hyperdynamic sepsis, Ang II restored arterial pressure, increased urine output and normalized creatinine clearance. This beneficial effect of Ang II supports the notion that sepsis‐induced acute renal failure is due to efferent arteriolar vasodilatation and a subsequent loss of glomerular filtration pressure. Ang II may be a safe and effective treatment to normalize arterial pressure and renal function in sepsis. Funded by NHMRC, Australia.
siRNA silencing of eotaxin‐3 (CCL26) modulates gene expression of eotaxin‐2 (CCL24) and CCR3 in alveolar type II cells
Authors: Bauer David M. , Taka Equar , Effahali Younes J. , Abonyo Barack O. , Thomas Ronald D. , Heiman Ann S.
Published: June 2021

Chronic diseases of the airway are characterized by an underlying inflammation. The CC chemokines CCL26 and CCL24 recruit and activate eosinophils (EOS) which participate in the airway inflammation. Our previous work demonstrated that CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of cytokines and chemokines in alveolar type II cells. Thus, we hypothesized that treatment of these cells with CCL26‐siRNA may decrease cytokine‐induced release of the eotaxins and suppress EOS activation. A549 alveolar type II cells were transfected with four CCL26‐siRNA duplexes then stimulated with IL‐4. ELISA results indicated >90% inhibition of IL‐4‐stimulated release of CCL26 (789±33.4 pg/ml) and CCL24 (206±2.31 pg/ml) in CCL26‐siRNA treated A549 cells. Densitometry results indicated a 64–71% decrease in CCR3 protein expression in siRNA treated cells and a >88% decrease in CCL26 mRNA as indicated by dotblots and RT‐PCR, respectively. Effects of siRNA treatments on EOS activation were then tested. Results indicated that CCL26‐siRNA treatment of alveolar type II cells reduced EOS superoxide anion generation by 70–90%. These findings demonstrate that siRNA technology may provide a new strategy for post‐transcriptional control of eotaxin gene expression, thereby ameliorating inflammation occurring in airway diseases. Supported in part by NIH grants RR08111 and RR03020.
Downregulation of ENaC expression and activity by Pseudomonas LPS in alveolar epithelial cells
Authors: Dagenais André , Morneau Frédéric , Boncoeur Émilie , Berthiaume Yves
Published: June 2021

Pseudomonas aeruginosa is a gram negative bacteria that causes chronic infection in cystic fibrosis patients and is known to modulate the epithelial Na channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested if LPS from P. aeruginosa could modulate the expression and activity of ENaC in alveolar epithelial cells. Alveolar epithelial cells purified from rat lung were cultured on permeable filters for 4 days before the cells were treated on the apical side with 15 μg/ml of Pseudomonas LPS. The treatment decreased gradually the total transepithelial current with the lowest point reached at 4h with a 35% decline compared to untreated cells. The ENaC (amiloride‐sensitive) portion of this current was declined by 53% at 4h. The α and βENaC mRNA were decreased respectively to 55% and 54% of control by a 4h LPS treatment. Estimation of ENaC activity at the apical membrane following amphotericin B permeabilisation of the basolateral membrane in presence of a Na gradient shows a 52% decrease in 4h LPS treated cells. The decline in ENaC current elicited by LPS is therefore the consequence of a decreased activity of the channel. Ro‐31 8820 a PKC inhibitor abolished the downregulation of ENaC activity in the cells and also restored the α and γENaC mRNA level back to normal. These results show that LPS downregulates ENaC mRNA expression and activity in alveolar epithelial cells by a PKC‐sensitive pathway.

Supported by CCFF and CIHR
Delayed lung expansion following pneumonectomy (PNX) improves gas exchange during exercise
Authors: Dane Dan Merrill , Yilmaz Cuneyt , Hsia Connie C.W. , Johnson Robert L
Published: June 2021

To examine post‐PNX response to expansion of the remaining lung separate from that to other signals (perfusion or inflammation), the right lung of adult male foxhounds was replaced by a custom‐shaped inflated silicone prosthesis filled to end‐expiratory lung volume (EELV) with 50/50 SF6/air. The prosthesis was acutely deflated after 4mo (DEF, n=6) or kept inflated (INF, n=3). Lung volume (mL.kg−1), pulmonary blood flow (Q̇c, mL·(min·kg)−1), lung and membrane diffusing capacities (DLCO and DMCO, mL·[min·mmHg·kg]−1), and pulmonary capillary blood volume (Vc, ml.kg−1) were measured at exercise pre‐PNX and 4 and 12mo post‐PNX. Exercise Q̇c was unaltered. At the same Q̇c (=400), DLCO and DMCO were 25–40% below normal 4mo post‐PNX. By 12mo in INF group, EELV, DLCO and DMCO further declined 28, 10 and 25%, respectively, while in DEF group these values were 87, 32 and 73% higher relative to INF group. Vc changed variably. Mean ± SD. P<0.05 *vs. normal, †vs. INF 4mo, ‡vs. INF 12mo. Thus, delayed lung expansion post‐PNX significantly improved gas exchange at exercise.

Supported by NHLBI R01 HL‐040070, 054060, 062873 and 045716.
Inhibition of endothelin‐induced response on equine bronchial rings
Authors: Venugopal Changaram , Holmes Earnestine , Beadle Ralph , Koch Catherine , Eades Susan
Published: June 2021

Airway hyperreactivity is the hallmark of human asthma and recurrent airway obstruction (RAO) in horses. Endothelin‐1 (ET‐1) is an inflammatory mediator that causes potent constriction of human and equine airways. Nuclear Factor‐kB (NF‐kB), a transcription factor, and Tumor Necrosis Factor‐alpha (TNFα), a cytokine, are well established molecules that are involved in inflammation. The objective of the study was to determine the effectiveness of Ammonium pyrrolidinedithiocarbamate (PDTC) and Pentoxifylline, antagonists of NFkB and TNFα respectively, on ET‐1 induced response of equine bronchial rings from RAO‐affected and unaffected horses. Rings from the 4th – 7th generation branches of airways were set in organ baths bubbled with O2 and filled with Tyrode's solution at 37°C. One side of the ring was fixed to the floor of the bath and the other end was connected to a force transducer interfaced with a polygraph. After application of two grams of tension and an equilibration period of 45 minutes, the response of the tissues to graded concentrations of ET‐1 was measured in one ring (control), one incubated with PDTC and another with Pentoxifylline. The results indicated that Pentoxifylline substantially inhibited the response caused by ET‐1 whereas, PDTC did not. These results suggested that TNFα may be involved in the contractile effects of ET‐1 in the equine airways with recurrent airway obstruction. Supported by Equine Health Studies Program of LSU School of Veterinary Medicine.
Low‐dose arsenic inhibits wound repair, upregulates MMP‐9 and alters Ca2+ signaling in human airway epithelial cells
Authors: Boitano Scott , Liguori Andrew E. , Olsen Colin E. , Zong Yue , Burgess Jefferey L. , Lantz R. Clark
Published: June 2021

The polarized conducting lung epithelium participates in innate immune defense in part by performing a barrier function to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung through inhalation or ingestion. We have recently shown that exposure of mice or humans to chronic low‐dose arsenic (10 – 50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report we use an in vitro model to examine effects of 24 hr exposure of low‐dose arsenic on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb slows formation of the epithelial monolayer in vitro, and inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we show that 24 hr arsenic exposure increases activity and expression of MMP‐9, an important protease in lung epithelial migration. Further, 24 hr arsenic exposure reduces purinergic‐dependent Ca2+ signaling following localized wound or direct application of ATP. We propose that low‐dose arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP‐9 and alteration of purinergic dependent intra‐ and inter‐cellular Ca2+ signaling by lung epithelial cells.
Raising Rural Student Achievement in Science Through Online Inquiry Based Teacher Professional Development
Authors: Bradshaw Timothy John , Cunningham Jessica , Shafer Sally , Bradley Kelly , Straley Joseph , Osborn Jeffrey
Published: June 2021

Teacher scientific content knowledge is critical to improving student achievement in science. Teacher engagement in inquiry based instruction improves long term student understanding of science. This study assessed changes in rural, middle school students’ understanding of standards based heat and temperature concepts after teacher professional development using an inquiry focused course offered by Newton's Universe (NU) distance learning program. Experimental students whose teachers trained with NU (n = 1072 students; 33 teachers) were compared to control students whose teachers had no NU professional development (n = 295 students; 5 teachers). A 39 multiple choice pre/post assessment quantified student heat and temperature understanding. Pre test scores were not significantly different between the two groups (control: 36.95 ± 10.43% correct; experimental: 36.57 ± 10.51% correct; p>0.05). Training with NU significantly increased the experimental group's scores to 46.03 ± 14.58% correct relative to pre test (p<0.05), while the control group did not change (37.27 ± 13.71% correct; p>0.05). Thus, teacher professional development by distance learning enhanced rural middle school students’ understanding of heat and temperature concepts. This model for higher education outreach to geographically isolated teachers may be effective for improving student achievement across scientific disciplines.
Teaching the cell based model of coagulation
Authors: Lorr Nancy , Catalfamo James
Published: June 2021

Coagulation reactions in vivo are localized to cellular surfaces. The endothelial cells that line the vascular space provide a non‐thrombogenic surface preventing unnecessary coagulation, thromboses and platelet activation. When this lining is disrupted, blood comes in contact with the extra vascular tissue exposing platelets to the platelet activating agent collagen. The activated platelet externalizes plasma membrane phosphatidylserines‐which provide a cell anchored scaffolding for formation of coagulation complexes, such as the prothrombinase and tenase complexes, accelerating coagulation reactions 300,000 fold. Activated platelets also form a platelet plug at the site of injury as they aggregate.

This laboratory exercise emphasizes the importance of cells in the coagulation process. Coagulation triggered by the “intrinsic” pathway is assayed by addition of inhibitors, Triton‐X, and EGTA to demonstrate the role of phosphatidylserine vesicles, and Ca2+ in the formation of the coagulation complexes. Thrombin conversion of fibrinogen to fibrin, is not inhibited by Triton‐X, demonstrating that the phosphatidylserine coagulation complexes are not required for this final step in the coagulation pathway

The activation and aggregation of platelets in response to collagen and ADP and their inhibition by prostaglandin E1 is monitored by changes in light transmission as the suspension of platelets undergo activation, first changing shape from disc to sphere and then as they form aggregates of various sizes.
Collaborative Group Testing Benefits High Performing As Well As Low Performing Students
Authors: Giuliodori Mauricio Javier , Lujan Heidi , DiCarlo Stephen Edward
Published: June 2021

We used collaborative group testing in a veterinary physiology course (65 students) to test the hypothesis that all students e.g. high performing and low performing students benefit from collaborative group testing. In the collaborative group testing format, students initially answered questions in the traditional format as individuals. Immediately after completing the exam as individuals, students answered the same questions in groups of two, and finally, the same questions were discussed by the instructor and the students. We measured two learning outcomes for every student, individual and group test scores, to assess the impact of collaboration on performance. Based on the results, group members were categorized as “high performing” and “low performing” and their scores were correlated with group scores. Finally, student evaluations of the format were collected. Student collaboration (group testing) enhanced performance (20%, P<0.05). Importantly, both high performing group members (5%) and low performing group members (38%) improved their scores (P<0.05). Furthermore, the group performance was strongly correlated with individual scores from both, high and low performing group members (R: 0.847 and 0.717, respectively, P<0.001). Finally, student evaluations to the collaborative group testing format were very positive. In conclusion, student collaboration during group testing was beneficial for high‐ and low‐performing group members.
Use of case studies and data analysis to improve critical thinking skills in a one‐semester anatomy & physiology course
Authors: Walton Kristen Lee Williams
Published: June 2021

One‐semester anatomy and physiology courses require students to learn an extensive amount of new vocabulary and concepts in a short period of time. Although critical thinking skills are important for learning any science subject, students often feel overwhelmed by content and chose memorization over learning in a way that promotes critical thinking about the subject. The use of case studies and problem‐based learning approaches is well‐documented to improve student engagement and retention. To provide students in a one‐semester anatomy & physiology course for allied health majors with opportunities to practice and develop critical thinking skills, case studies and data analysis activities were used as in‐class or take‐home assignments. Case studies and data analysis activities were written by the instructor or drawn from the National Center for Case Study Teaching in Science or articles on teaching with classic papers in physiology. Many students demonstrated an improved ability to work through complex physiological problems, such as applications of homeostasis, by the end of the semester. Students were surveyed for self‐assessment of the value of these activities in increasing their understanding of content and concepts. The addition of activities that require students to analyze and apply content to case studies or data can increase student learning and critical thinking in anatomy and physiology.
Utilization of an interdisciplinary case study on vision to enhance student learning at the interface of biology and physics
Authors: Barney Christopher C. , Mader Cathy , DeWeerd Alan
Published: June 2021

As undergraduates become the biomedical research scientists of the future, they will need to be prepared to work as members of interdisciplinary teams. At Hope College, we are working to better prepare our undergraduates for this future by incorporating interdisciplinary case studies in our science courses. One such case study is on the physics of vision, which is being used in introductory biology and physics classes. Students are asked to compare the focusing systems of the nautilus, scallops, and humans by using optic benches to explore the physics of pinholes, mirrors and lenses. In addition, students are asked to make recommendations on ways in which visual acuity, light sensitivity, or color range can be enhanced using a genetic engineering approach. Students demonstrated a good understanding of the physics and biology of vision in their reports and exams. Students reported significant gains in confidence in their ability and willingness to approach problems from more than one scientific perspective, in their appreciation of the importance of physics in understanding biological functions, and in their awareness of the complexity of scientific problems. Interdisciplinary case studies have the potential to help better prepare our students for future work as biomedical scientists but require a substantial time investment by faculty and a willingness to work outside of one's area of training. Supported by HHMI.
Using General Models in Physiology can Enhance Student Understanding
Authors: Wenderoth Mary Pat , Henson Kate
Published: June 2021

Students often compartmentalize their knowledge and fail to recognize the principles that are common between topics. General models (GM) are teaching models that provide a way of making common physiological patterns explicit to students (Modell, Adv. Physiol. Educ. 23:101–107, 2000.) We predicted that by introducing and incorporating the concepts of GM into an introductory physiology course, students could use the GMs to form robust mental models that would help them explain, predict and successfully solve physiological problems. Four of the seven GMs: Flux, Mass Balance, Mass Action and Control Systems were presented. Exams in the course are short answer and students were directed to identify the GM in the margin when relevant. We recorded if students used the correct GM and the points scored less GM pts. Students who correctly identified GMs earned significantly more points on exam questions. Prior to beginning the Respiratory System, students had a pre‐test on the mechanics of ventilation. No prompts to use GM were given, yet 90% of students used the correct GM to explain the mechanism. We also analyzed student's perception of the usefulness of GM and found that students who correctly used GM also had the most sophisticated understanding of the usefulness of GM for their explanatory and predictive powers. Using GM in your course has the potential to significantly enhance student understanding in physiology.
Additional evaluation of the value of students teaching students (STS) in gross anatomy laboratory
Authors: Lee Vaughan H , Vidic Branislav
Published: June 2021

In previous reports the STS system in the medical gross anatomy laboratory was explained and discussed relative to effectiveness of students mastering anatomical learning objectives. In 2006 the system was expanded to its current form where approximately one half of laboratory sessions were STS, while the others were classically presented. There was no difference in academic success between the two methods as attested by performance on local and national examinations. This past year we evaluated additional professional benefits which included promoting self‐directed learning, emphasizing independent location and retrieval of pertinent information, differential reasoning, and communication and teamwork among students. At the end of the 2007 course, a voluntary survey among students was conducted to determine whether the STS exercises helped improve the skills and awareness of aforementioned properties. An overwhelming majority of students surveyed (74–100%) felt these skills and behaviors were enhanced by the STS exercises. Only 60% of students felt that the subject of professional ethics was sufficiently incorporated in the STS system. Our future goal is to augment the STS system in order to improve the student's awareness and attitudes of professionalism that will be required throughout their medical education and life as physicians. Supported by TTUHSC Dept. of Cell Biology and Biochemistry.
Variability in the location of the mandibular foramen in African‐American and Caucasian populations of male and female skulls
Authors: Bee Mary Tracy , Rabban Milad , Sethi Herpreet , Tran Tracy , Baker Chad , Forbes Bill
Published: June 2021

The mandibular foramen is a hole on the medial surface of the mandible of humans, through which the inferior alveolar nerve passes. This nerve is targeted when anesthetizing the lower jaw, as required in many dental procedures. Our research investigated variability in the position of the mandibular foramen in male and female populations, as well as in Caucasian and African‐American populations. One hundred skulls from the Hamaan‐Todd collection at the Cleveland Museum of Natural History were digitally photographed and analyzed. Preliminary results do not identify a significant difference in the position of the foramen between males and females (p>0.05). However, a significant difference in the antero‐posterior position of the foramen in Caucasian and African‐American populations was identified (p<0.01). This has great clinical relevance as it may result in variable treatment and positioning of anesthesia needles in patients of different races.
The nasal capsule of platyrrhine primates in relation to paranasal pneumatization
Authors: Smith Timothy DeWolf , Docherty Beth , Rossie James , Bonar Christopher , Burrows Anne
Published: June 2021

Recent work posits that “true” paranasal sinuses form by an invasive process that is distinct from general bone remodeling. One hypothesis holds that breakdown of the cartilaginous nasal capsule that surrounds recesses is a necessary prelude to secondary pneumatization. To test this hypothesis, we examined heads of perinatal New World primates (n = 9) that were serially sectioned in the coronal plane. In two species that form frontal sinuses that derive from the frontal recess, the lateral wall of the nasal capsule (paries nasi) is relatively less extensive compared to three species in which no frontal sinus forms from this recess. A single species in our sample (Saimiri sciureus) does not form a maxillary sinus. This species is distinguished by the presence of extensive remnants of the inferior margin of the paries nasi. More generally, in species with postnatal maxillary or frontal sinuses, the nasal capsule along the wall of the maxillary or frontal recesses is more fragmented. These findings support the hypothesis that nasal capsule breakdown is a crucial event preceding sinus formation. These findings also suggest that absence of “true” sinuses may correlate with protracted perinatal presence of capsular cartilage surrounding the natal recess.
Palatal Pneumatization via the Maxillary Sinus: A Comparative Anatomical Study of Arboreal Primates and Terrestrial Mammals Employing CT imaging and Gross Dissection.
Authors: Osbourne Aton , Delson Eric , Marquez Samuel
Published: June 2021

Palatal pneumatization (PP) via the maxillary sinus (MS) is a common occurrence in mammalian species but rare in humans. This study tracks the frequency and distribution of such pneumatization, as the maxillary sinus is ubiquitous throughout Mammalia and is considered a primitive eutherian condition. This study examined catarrhine primates (great apes ‐ gorillas, chimp, and orangutans ‐ and Old World monkeys), and a diverse array of terrestrial mammals including bear, deer, mountain goat and cow. Humans were included in our study to serve as an out‐group comparison. Results from CT imaging and gross anatomical dissection revealed that gorilla, orangutan, Old World monkeys, and cow exhibit a maxillary sinus but do not present a pneumatized hard palate. PP was present in deer, bear, mountain goat, chimpanzee and modern humans. The presence and extent of MS pneumatization into the hard palate might be hypothesized as related to the dispersal of forces generated by biomechanical stresses and strains usually associated with masticatory behavior. However, lack of dietary consistency within the two groups of species (with and without pneumatization) tends to reject this hypothesis. Interestingly, chimpanzees and humans are the only hominoids which exhibit PP, and because these species are sister taxa (each others’ closest relative), we suggest that this craniofacial feature was present in their common ancestor.
Integrin engagement reduces APE1 co‐immunoprecipitation with XRCC1 in hydrogen peroxide‐treated murine lung endothelial cells
Authors: Stevens Rachel L. , Hoyt Dale G.
Published: June 2021

Reduction of genotoxin‐induced DNA 3’OH end labeling and histone 2A.X (H2A.X) phosphorylation after integrin engagement requires certain Base Excision Repair (BER) proteins; however, integrin engagement does not reduce the amount of physical DNA breaks as measured by the comet assay. Consequently, we hypothesized that integrin signaling mediates the outcome of specific processing events during the BER response. To address this, murine lung endothelial cells were plated overnight in DMEM with 0.5% FBS and treated with 1 μg anti‐β1 integrin antibody/ml or the corresponding non‐immune IgG for 1 h followed by addition of 2 μg goat anti‐rat secondary antibody/ml for 4 h. Cells were then treated with and without 0.001M H2O2 for 15 min. to induce DNA damage. XRCC1 was successfully immunoprecipitated (IP) after incubating cell lysate with XRCC1 antibody overnight. Subsequent Western blotting for APE1 revealed that the non‐immune IgG‐treated samples damaged with H2O2 exhibited the expected increase in APE1‐XRCC1 association by IP. By contrast, integrin engagement significantly reduced co‐IP of APE1 with XRCC1 after H2O2‐induced damage. These results suggest that integrin signaling modulates the response to DNA breakage through altered BER protein interactions, and may account for the previously observed reductions in DNA 3’OH and H2A.X phosphorylation.
Identification and Characterization of Protein‐Protein Interactions between Yeast Rap1p and the TFIID Complex
Authors: Layer Justin Harrison , Weil Peter Anthony
Published: June 2021

In yeast transcription of Ribosomal Protein (RP)‐encoding genes is heavily dependent upon the TFIID complex, composed of the TATA Binding Protein and 14 associated Tafps. RP gene transcription is also directly influenced by the DNA binding transcription factor Repressor Activator Protein (Rap1p). Recently we showed that Rap1p and TFIID engage in direct interaction, this is important for transcription of RP genes. We have used Far Western Protein Blotting coupled with truncation mutagenesis to identify the regions responsible for Rap1p‐TFIID interaction in all three Tafp subunits involved. Rap1p interacts with Taf4p through the conserved region between the canonical Histone Fold Domain (HFD) and the Concerved C‐Terminal Domain (CCTD). Rap1p contacts Taf5p through residues located in the highly conserved NTD regions. We confirmed regions involved are essential for cellular viability in both Taf4p and Taf5p. Consistent with this observation, using random mutagenesis we were able to generate large families of conditional mutants of both TAF4 and TAF5 linked to regions encoding specific domains of the proteins. We are now using these tools in systematic molecular genetic and biochemical dissection of this interaction. This study will allow us to characterize Rap1p‐TFIID interaction in much greater detail.
Quantitative regulation of gene expression of human endothelial tumor markers by thrombospondin‐1 and nitric oxide crosstalk
Authors: Li Dayan , Isenberg Jeff S. , Pendrak Michael L. , Roberts David D.
Published: June 2021

Angiogenesis, growth of new blood capillaries from existing vessels, is essential for progression of solid tumors. Thrombospondin‐1 (TSP1) is one of the most potent angiogenesis inhibitors and regulates nitric oxide (NO) signaling, which can be both inhibitory and stimulatory for blood vessel formation. Expression of several known tumor endothelium marker genes including extracellular matrix collagen Iα1, Iα2, IIIα1 genes, basement membrane collagen IVα1, laminin‐α3, and laminin‐α4 genes, and TEM8 was analyzed using quantitative RT‐PCR in human umbilical vein endothelial cells (HUVECs). Compared to untreated HUVEC and normalized to mRNA levels for the housekeeping gene hypoxanthine‐guanine phosphoribosyl transferase, mRNA levels of all of the tumor marker genes were downregulated 40 to 85% and 37 to 78% by exposure to exogenous TSP1 (2.2 nM) and NO donor DETA/NO (10 μM), respectively. When HUVECs were treated with both TSP1 and NO, mRNA expression of collagens Iα2, IIIα1, IVα1, laminin α3 and α4, and TEM8 rebounded toward control levels, indicating cross talk between TSP1 and NO in the coordinate regulation of these tumor endothelial markers.
Identification of a potential role of NPM in transcriptional regulation
Authors: Yung Benjamin Yat Ming , Liu Hsuan , Tan Bertrand Chin Ming , Chiu Ya Ming
Published: June 2021

Nucleophosmin/B23 (NPM) is an important nucleolar phosphoprotein with pleiotropic functions in various cellular processes. Its altered expression in tumor cells indicates an intimate link of this protein to tumorigenesis. Exact mechanism underlying NPM‐mediated cell growth control remains largely elusive. Our recent findings have identified a potential but mostly uncharacterized role of NPM in transcriptional regulation. We found that AP‐2α forms complex with NPM during retinoic acid (RA)‐induced cell differentiation. AP‐2α recruits NPM to the promoters of certain RA‐responsive genes, such as p120, Hsp60 and b‐Myb, and such binding is concomitant with their transcriptional repression. RNAi suppression of NPM alleviates the negative regulation of gene expression exerted by RA treatment. Furthermore, we found that NPM binds to the initiation site of PCNA promoter. C‐terminal site of NPM is critical in activation of PCNA promoter. Acetylation mutations at c‐terminal block NPM‐activated promoter activity. Acetylated NPM mutants that could not activate PCNA promoter are not recruited to the promoter. NPM enhances acetylation‐dependent PCNA transcription in conjunction with histone H4 acetylation. These studies underscore the functional significance of NPM in controlling cell growth and differentiation, and delineate a novel mechanism by which nucleophosmin/B23 may exert such regulatory role.
Kinetic analysis of template competitive inhibitors of reverse transcriptases from HIV‐1, MMLV, and AMV
Authors: Lin Tao , Doughty Michael B.
Published: June 2021

Many viral diseases threaten people's health and there are too few vaccines or viral inhibitors to fight against these viruses. One example is the HIV retrovirus. HIV encodes the enzyme reverse transcriptase (RT) that converts single‐stranded RNA genome of the retrovirus into double‐stranded DNA before the cell begins viral replication. The goal of our research has been to synthesize nucleotide inhibitors that can directly inhibit the activity of RT and prevent viral replication. Four inhibitors molecules synthesized and characterized kinetically are: 1) 2‐S‐(3,4‐dichlorobenzyl)‐etheno‐dATP, 2) 2‐S‐(4‐chlorobenzyl)‐etheno‐dATP, 3) 2‐S‐benzyl‐etheno‐dATP, and 4) 2‐S‐(4‐methoxybenzyl)‐etheno‐dATP. Five component inhibition assays were performed, either as IC50 or Ki plots, against three enzymes, HIV‐1, MMLV, and AMV reverse transcriptases. The results of enzyme assays performed to test the inhibition of HIV RT will be presented.
Induction of apoptosis by novel substrate selective extracellular signal‐regulated kinase (ERK) inhibitors
Authors: Smith Sarice R , Shapiro Paul
Published: June 2021

The ERK proteins are potent mediators of cell proliferation and survival. Unregulated activation of the ERK proteins plays a role in the progression of a variety of cancers. Thus, targeted inhibition of ERK's function in promoting cell proliferation and survival is viewed as a promising approach for anti‐cancer therapy. Using in silico modeling, we have recently developed small molecular weight compounds that target ERK domains involved in specific substrate interactions. Compounds designed to target the common docking (CD) domain on ERK2 were tested for their ability to block anti‐apoptotic mechanisms involving ERK phosphorylation of p90RSK‐1, which promotes cell survival by phosphorylating and inactivating the pro‐apoptotic protein BAD. HeLa cells treated with 50 μM of one compound (termed 76) caused induction of apoptotic signaling pathways as measured by cleavage of poly (ADP‐ribose) polymerase (PARP), which was observed within 3 hours. PARP cleavage induced by 76 required activation of the intrinsic apoptotic pathway, specifically caspase 9 and caspase 3. Compound 76 inhibited p90RSK‐1 phosphorylation of BAD in a manner that correlates with caspase activation. Furthermore, inhibition of BAD phosphorylation in the presence of compound 76 was not due to off‐target effects on other pro‐survival pathways. These findings suggest that substrate selective inhibition of ERK signaling promotes apoptosis in transformed cells, illustrating a novel approach to the development of anti‐cancer therapies. (Supported by NIH CA120215)
Zinc inhibition of bovine phenol sulfotransferase (bSULT1A1)
Authors: Lapham David E. , Beckmann Joe D.
Published: June 2021

Sulfotransferases (SULTs) catalyze sulfate transfer from 3'‐phosphoadenosine‐5'‐phosphosulfate (PAPS) to various acceptors such as phenols and steroids. Zinc is known to inactivate SULTs; however, the mechanism of this inhibition is unknown. We investigated this question using purified recombinant bovine enzyme (bSULT1A1), which reacts broadly with phenolic substrates. Enzyme activities were determined by continuous recording of fluorescence decay during 7‐hydroxycoumarin sulfation. Greatest inhibition was achieved by addition of zinc prior to the substrates, suggesting formation of a SULT:zinc complex. Inhibition is partially reversed upon addition of 2‐mercaptoethanol or glutathione. Zinc dose‐response curves revealed IC50 values of approximately 9 μM at pH 7.4, compared to 270 μM at pH 5.5. The zinc pH inhibition curve suggests the involvement of a titratable group of pKa ≈ 6.5. Detailed initial velocity analysis with varied PAPS and zinc concentrations indicated zinc to be a non‐competitive inhibitor with a Ki = 13.6 μM. Isothermal calorimetric titrations with zinc were adequately fit with a one‐site model and yielded a Kd of 2.47 μM with a stoichiometery of 2 metals per bSULT1A1 subunit at pH 7.4. The sensitivity of bSULT1A1 to zinc inhibition, and the reversibility of the inhibition by glutathione, raise questions regarding the role of this metal in regulating sulfation of hormones and drugs.
Slow Onset Inhibition of KasA by Thiolactomycin: Mechanistic Insights for Lead Optimization
Authors: Machutta Carl , Tonge Peter
Published: June 2021

Mycobacterium tuberculosis (MTB) is a global health threat and novel chemotherapeutics are urgently needed for the treatment of multi‐drug resistant and extensively drug resistant strains. In our search for novel TB chemotherapeutics we are focusing on KasA, the β‐keto‐acyl ACP synthase enzyme in the type II fatty acid biosynthesis pathway (FASII), which is a validated target for drug discovery. KasA, which catalyzes a Claisen condensation to initiate each round of fatty acid elongation, is inhibited by the natural product thiolactomycin (TLM). In order to direct the synthesis of more potent TLM analogues, we are using fluorescence spectroscopy and enzyme inhibition studies to analyze the interaction of TLM with KasA. Inhibition studies of ketoacyl synthase enzymes by TLM routinely include a preincubation step prior to substrate addition. We have shown that while TLM binds relatively weakly to the free enzyme, the compound is a slow onset high affinity inhibitor of the KasA acyl‐enzyme intermediate. Currently we are extending these studies to C171Q KasA, a mutant thought to mimic the acyl enzyme intermediate. In parallel experiments, interligand‐NOE NMR studies are being used to optimize the binding of TLM to KasA. These data are being used to guide the synthesis of additional TLM analogs.
Predicting gene dosage using genomic sequence data
Authors: Barker Jocelyn Elaine , Sherlock Gavin , Hartman James , Morgan William
Published: June 2021

Copy number variation (CNV) has been shown to have important phenotypic consequences and occurs in organisms as diverse as yeast and man. We wish to create a pipeline that compares genomic sequence reads with a reference genome sequence to predict regions with copy number variation (including deletions). We are using three approaches. The first approach assumes that the number of sequencing reads for a genomic region follows a Poisson distribution. Based on the observed number of reads aligning to a region, we can then determine a 95% confidence interval for the actual copy number. The second method identifies sequence reads that partially align at two locations in the reference genome. Trivially, such reads could be chimaeric, but alternatively, this could indicate sequence duplications or deletions. In the case of a duplication, we would expect at some frequency (depending on sequence coverage) to also observe reads with that sequence in its normal location within the reference sequence. The final approach, which is currently in progress, examines the length of continuous regions along the reference sequence that either are or are not represented in the sequence reads, and assigns a probability based on this occurring by random chance. Regions for which this probability is low indicate a probable duplication or deletion, respectively. To develop and test our methodology for predicting CNV regions, we are analyzing data from the baker's yeast S. cerevisiae, using the high quality reference sequence and sequencing data from 37 additional strains. Subsequently, the phenotypic variability of the strains under different growth conditions will be examined and correlated to the proposed copy number to investigate possible functional consequences of the observed copy number variation.
Adrenocorticotropin‐Stimulated Phosphorylation of RhoA Facilitates Binding to mDia1, Mitochondrial Trafficking, and Cortisol Biosynthesis
Authors: Li Donghui , Sewer Marion B
Published: June 2021

Steroid hormones are formed by several successive enzymatic transformations of the substrate cholesterol in the mitochondria and endoplasmic reticulum (ER). In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and andrenal biosynthesis by activating a cAMP/cAMP‐dependent protein kinase (PKA) pathway. In this study, we used the H295R human adrenocortical cell line to show that ACTH and Bt2cAMP increase mitochondrial movement. Agents that alter the polymerization of tubulin attenuate both mitochondrial movement and cortisol biosynthesis. The interaction between RhoA, one of member of the Rho family of small GTPases and one of its effectors, mDia1 regulate ACTH/cAMP‐stimulated mitochondrial trafficking and cortisol production. Phosphorylation of RhoA in response to ACTH increased the binding of RhoA to mDia and facilitated both mitochondrial movement and cortisol secretion. We conclude that this increase in organelle trafficking facilitates cortisol production by bring mitochondria and ER in close proximity for efficient transfer of substrate.
Kdo2‐Lipid A stimulated GIVA Phospholipase A2 catalyzed AA release is Phosphatidic Acid Phosphohydrolase‐1 Dependent
Authors: Grkovich Andrej
Published: June 2021

Macrophages respond to toll‐like receptor 4 (TLR‐4) agonists, such as LPS and Kdo2‐Lipid A, by upregulating cyclooxygenase 2 (COX‐2) and releasing prostaglandins. Group IVA phospholipase A2 (GIVA PLA2) catalyzes the deacylation of membrane‐bound arachidonic acid (AA) from the sn‐2 position of glycerophospholipids, which can then be further metabolized by COX‐2 in the synthesis of prostaglandins. Phosphatidic acid phosphohydrolase‐1 (PAP‐1) is a Mg+2 dependent, membrane‐associated protein involved in the synthesis of triacylglycerol. We show herein that GIVA PLA2‐mediated AA release and COX‐2 expression through TLR‐4 signaling mechanism requires PAP‐1. PAP‐1 chemical inhibition decreased COX‐2 mRNA transcript production, COX‐2 protein expression and AA release from macrophages. Addition of diacylglycerol, the end‐product of PAP‐1 catalysis, enhanced AA release from uninhibited cells and restored the AA release in inhibited cells. TLR‐4 activation increased the levels of endogenous cellular DAG within two minutes of stimulation and this increase was sensitive to PAP‐1 inhibition. Furthermore, PA phosphohydrolase activity assays showed that PAP‐1 activity translocated from the cytosolic fraction to the membrane fraction within two minutes of stimulation. DAG add‐back experiments demonstrate that TLR‐4 induced COX‐2 expression is enhanced by the addition of exogenous DAG. Additionally, PAP‐1 chemical inhibition reduced the synergistic release of AA from cells stimulated with TLR‐4 agonist and ATP, while not affecting the magnitude of synergistic activation.
Oxidative Stress Induced by Free Iron Stimulates Phosphatidylcholine Breakdown in Cerebral Cortex Synaptic Endings
Authors: Mateos Melina Valeria , Salvador Gabriela Alejandra , Giusto Norma Maria
Published: June 2021

Neuronal damage induced by transition metal ions is comparable with the toxic effect of β amyloid peptide. Our purpose was to study the effect of oxidative insult on diacylglycerol (DAG) generation from phosphatidylcholine (PC) in rat cerebral cortex synaptosomes. DAG from PC can be generated by phospholipase D (PLD) and phosphatidic acid phosphatase 2 pathway or by a phosphatidylcholine‐specific phospholipase C (PC‐PLC).

Oxidative injury induced by FeSO4 increased DAG generation by 77% with respect to control conditions. Experiments carried out in the presence of ethanol, demonstrated that both PLD and PC‐PLC pathways contributed to DAG generation in the presence of iron. Additionally, free fatty acid generation was inhibited in the presence of Fe2+. Neither the specific phosphoinositide‐3‐kinase inhibitor (LY294002) nor Herbimycin A modified the DAG increase induced by Fe2+. Assays conducted with Fe2+ and either DAG lipase or DAG kinase inhibitors, showed no differences in DAG levels. The phosphoinositide‐PLC inhibitor, U73122, inhibited DAG formation by 23% after 60 min of iron exposure.

Our results demonstrate that the generation of lipid messengers through PC breakdown is activated under oxidative injury conditions in synaptic endings.

Supported by Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional del Sur and Agencia Nacional de Promoción Científica y Tecnológica.
Cholesterol Transport Properties of NPC2 Protein: In Vitro Analysis Using Stopped Flow Fluorescence Spectroscopy.
Authors: Xu Zhi , Farver William , Storch Judith
Published: June 2021

NPC2 is a soluble intralysosomal protein that binds cholesterol in vitro. To determine its potential role as a cholesterol transport protein, several fluorescence‐based assays have been developed. Cholesterol transfer from NPC2 to model phospholipid membranes is monitored by dequenching of the endogenous tryptophan fluorescence of NPC2, and cholesterol transfer from membranes to NPC2 is followed by monitoring the quenching of NPC2 tryptophan fluorescence by cholesterol. Cholesterol transport between membranes is also studied by utilizing dehydroergosterol (DHE), a fluorescent analog of cholesterol, and a fluorescence resonance energy transfer (FRET) assay. The results show that the transport of cholesterol by NPC2 protein occurs very rapidly via a collisional mechanism which involves direct NPC2‐membrane interactions, and which is markedly affected by membranes phospholipid composition. A series of point mutations that neutralize charged residues on the surface of NPC2 are being used to analyze the structural determinants of NPC2's cholesterol transport properties. For several mutants examined (K32A, D72A, K75A), cholesterol binding is unaffected yet cholesterol transfer is blocked. These studies support a role for the NPC2 protein in the egress of LDL derived cholesterol out of the endosomal/lysosomal compartment.