bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary supplementation with cholesterol and docosahexaenoic acid (DHA) affects cerebral omega‐3 to 6 fatty acid ratio and learning behavior of young pigs
Authors: Kim Sung Woo , McGlone John J. , Pond Wilson G. , Smith Stephen B. , Wu Guoyao
Published: June 2021

The ontogeny of brain development in the pig resembles that of humans more than any other non‐primate mammal. Cholesterol (chol) and DHA are essential constituents of brain cells. The aim of the research was to determine the effects of dietary DHA and chol and their interactions on cerebral fatty acid composition, and learning behavior in the neonatal pig. Sixteen newborn pigs were nursed by sows for 24 h and then assigned to 4 treatment groups, representing supplementation with chol (0.0 or 0.2%), and DHA (0.0 or 0.2%) to the basal formula mainly consisting of coconut oil, soybean oil, soybean flour, whey, and whey protein. Pigs were housed individually and fed ad libitum for 7 wk. Body weight and food intake were measured weekly. At age 21, the pigs were exposed to the Morris water maze test. All piglets were euthanized at d 49 to obtain cerebrum for determining fatty acid composition. During the 7 wk period, weight gain and food intake did not differ among treatments. Pigs without chol and DHA required longer (P < 0.01) time to complete the water maze than pigs in other treatments. Cerebrum of pigs with both chol and DHA had greater (P < 0.05) omega‐3 to 6‐fatty acid ratios than pigs in other treatments. Weight of cerebrum did not differ among treatments. This study indicates that dietary DHA and chol increase cerebral omega‐3‐fatty acid contents and beneficially enhance the learning ability of young pigs.
Predictors of Weight Loss in Mexican American Adolescents
Authors: Johnston Craig A , Tyler Chermaine , Foreyt John P
Published: June 2021

This study examined predictors of weight change in Mexican American adolescents. 80 overweight Mexican American children were randomized to receive either the intensive intervention or self help program. Physiological (e.g. standardized BMI (zBMI), percent body fat, and tanner stage), psychological (e.g. weight dissatisfaction, and health related quality of life), and behavioral (e.g. change in zBMI at 3 months) variables taken at baseline were examined. A stepwise regression was conducted to determine predictors of weight loss at 6 months and accounted for 57.1% of the variance in explaining weight change ( p<.001). Significant predictors of change in zBMI at 6 months included: zBMI at baseline (β= ‐.11, p<.05), change in zBMI at 3 months (β=.70, p<.001), and psychosocial functioning (β=‐.10, p<.05). Change in zBMI at 3 months accounted for 44% of the variance, while psychosocial functioning, and zBMI at baseline accounted for 3.9, 5.4 and 4.1% of the variance, respectively. Overall, the behavioral predictors accounted for the largest amount of the variance in explaining change in zBMI at 6 months. While psychological and physiological variables were significant predictors of weight change, behavioral changes may be the most important predictor. Psychological variables were unrelated to weight change.
Ac‐SDKP prevents cardiac fibrosis and dysfunction induced by galectin‐3, role of TGF‐β/Smad3 signaling pathway
Authors: Liu Yun‐He , D'Ambrosio Martin , Liao Tang‐Dong , Peng Hongmei , Rhaleb Nour‐Eddine , Sharma Umesh , Carretero Oscar A
Published: June 2021

We hypothesized that Ac‐SDKP (N‐Acetyl‐Seryl‐Aspartyl‐Lysyl‐Proline) prevents galectin‐3 (Gal‐3) induced cardiac inflammation, fibrosis and dysfunction, and these effects are mediated by the TGF‐ β/Smad3 signaling pathway. We infused Gal‐3 or/and Ac‐SDKP intrapericardially. Adult male Spregue Dawley rats received the following treatment for 4 weeks: vehicle, Ac‐SDKP (800 µg/kg/day), Gal‐3 (0.5 µg/hr), and Ac‐SDKP + Gal‐3. Hemodynamics, left ventricular ejection fraction (LVEF) and transmitral velocity were measured by echocardiography; inflammation and collagen deposition in the heart by histological and immunohistochemical staining and TGF‐β expression and Smad3 activation (p‐Smad3) by Western blot. We found 1) Gal‐3 enhanced macrophage and mast cell infiltration, increased cardiac interstitial and perivascular fibrosis; decreased ‐dP/dt response to isoproterenol challenge, E/A ratio and LVEF. 2) Ac‐SDKP prevented these adverse effects; 3) Gal‐3 increased TGF‐β expression by 3.94 fold (p < 0.01, Gal‐3 vs vehicle) and Smad3 phosphorylation 5.66 fold (p < 0.05, Gal‐3 vs vehicle) in LV; Ac‐SDKP reduced TGF‐β to 1.2 fold (p < 0.05); p‐Smad to 1.3 fold (p = 0.052), Gal‐3 + Ac‐SDKP vs Gal‐3). We conclude that Ac‐SDKP prevents Gal‐3 induced cardiac inflammation, fibrosis and dysfunction possibly via inhibition of the TGF‐β /Smad3 signaling pathway.
Adipocytes, PAI‐1 and MCF‐10A cell motility
Authors: Carter Jennifer , Church Frank
Published: June 2021

In addition to its role as energy stores, adipose tissue has been identified as an endocrine organ in recent years. Our understanding of the role of adipose in breast cancer development is limited, though obesity is an identified risk factor. In this study, we investigated the expression of plasminogen activator inhibitor type‐1 (PAI‐1) and urokinase plasminogen activator (uPA) in cells isolated from both omental and breast adipose tissues. uPA was not expressed in cells from either fat depot. In omental cells, PAI‐1 was more highly expressed in the preadipocytes. In cells from the breast, PAI‐1 was only expressed in the adipocytes. These results are of interest because expression of PAI‐1 is linked to poor patient survival in breast cancer. We also studied the role of breast adipocytes in breast epithelial motility, as we have seen an increase in phospho‐AKT in these cells when treated with breast adipocyte CM. We treated normal MCF‐10A cells with conditioned media (CM) from breast adipocytes and preadipocytes. Using the modified‐Boyden chamber model, we see a 2‐fold increase in MCF‐10A motility when treated with adipocyte CM, compared to preadipocyte CM. We hypothesize factors secreted from breast adipocytes, possibly PAI‐1, are inducing a more motile phenotype in the cells; further studies are needed to delineate the mechanism. These data provide a potential link between obesity and breast cancer development.
Regulation of Substrate Processing by the Proteasome
Authors: Finley Daniel
Published: June 2021

The proteasome, a large protease, recognizes the target protein via its ubiquitin chain, unfolds the protein, removes the chain, and injects the unfolded protein through a narrow gated channel into the large interior chamber of its 28‐subunit core particle, where it will be degraded.

Ubiquitin‐dependent protein degradation is essential for cells to survive many environmental stresses. Stress regulates the proteasome by changing its composition. In one example, we identified a response to deficient ubiquitin levels, or "ubiquitin stress." Ubiquitin stress does not upregulate proteasomes. Instead, ubiquitin stress alters proteasome composition. The gene encoding deubiquitinating enzyme Ubp6, which associates with the proteasome, is induced when ubiquitin levels fall. Enhanced loading of proteasomes with Ubp6 alters proteasome function. Ubp6 spares ubiquitin from proteasomal degradation via its deubiquitinating activity and probably also because of its capacity to inhibit the proteasome noncatalytically. This pathway, originally described in yeast, seems to function comparably in mammals.

Ubp6 works in tension with the proteasome‐associated ubiquitin ligase Hul5. Ubp6 attenuates chains while Hul5 extends them. When Hul5 is missing, the proteasome works with impaired fidelity, perhaps because chain extension is needed to prolong the substrate's association with the proteasome. Unlike Ubp6, Hul5 is not induced by ubiquitin stress. Thus, when proteasomes are remodeled under some stress conditions, the ratio of Ubp6 to Hul5 is changed.
Use of Virtual Anatomy and Physiology Laboratory Exercises: Evaluation of Student Learning
Authors: Kingsbury Jeff , Haberstroh Paul
Published: June 2021

Assessment of student learning in anatomy and physiology is an important aspect of science curriculum in undergraduate and graduate education. The use of computer models for anatomic instruction and physiology exercises is increasingly popular for a variety of reasons (logistical, cost, convenience, etc). There remains legitimate concern, and debate, about the effectiveness of this modality as evidenced by learning outcomes. This study continues previous work where we have examined student experiences objectively (student performance), and subjectively (student assessment) comparing virtual and traditional dissection modalities of education. Student evaluation records and exam performance records were recorded over a nine semester period. These were divided into two groups: group 1 participated in primarily traditional methodology (cadaver dissection, physiology lab exercises) and group 2 participated in primarily virtual lab exercises. The mean class test scores in group 1 was 74.7%, and group 2 was 78.8%. The average student evaluation scores were 2.92/3.00 in group 1, and 2.98/3.00 in group 2. By the Student "t" test on the binomial distribution, student performance was statistically different among the two groups when measured objectively, or assessed subjectively. These results suggest that use of computerized models and exercises is as effective as traditional use of cadavers in the undergraduate setting, when measured by student achievement of learning outcomes, and student's subjective perception of the experience.
The NIH supported Virtual Human Embryo (VHE) Project: educational and research resources for human embryology
Authors: Cork R. John , Gasser Raymond F.
Published: June 2021

The Carnegie Collection that is housed at the Human Developmental Anatomy Center (HDAC) in Washington DC is considered by many to be the best collection of human embryos in the world. The overall aim of the VHE project that began in 2000 is to make the best representative examples from each of the 23 Carnegie stages available to embryology researchers and educators, either on disks or online. To date, we have completed databases for 20 embryos (stages 1 ‐ 15 & 23) and are presently working on stages 16, 17, 18 & 20. The project will be completed within the next two years depending on continued funding from NIH. Each of the databases includes: a complete set of labeled sections viewable at three or four levels of magnification, a 3D computer model of the embryo, animations and other documents and figures related to the specimen. The disks have recently been revised to include an overview of each stage, a consistent format and the most current terminology for labels. We will show how these resources are freely available for educators to incorporate into presentations and/or class work and will give some examples of how the databases are being used for research into human embryology.

Supported by NIH grants RO1 HD 37811 and RO1 LM 7591
Seven Year Experience Using an Annotated Key Drug List in Teaching Pharmacology.
Authors: Mokler David J. , Reese Richard E.
Published: June 2021

There are > 6,000 prescription drugs available in the US. In the past 7 years we have used annotated, clinically relevant Key Drug Lists in the pharmacology portion of each System for second‐year medical students. These lists, updated annually, are generated by the pharmacology faculty with input of active primary‐care and subspecialty clinicians, PharmDs, and evidence‐based medicine data from the medical literature, including national treatment consensus guidelines, national listings of top drugs, Medical Letter's Guidelines, etc. Annotations for each drug include mechanism of action, major indication(s) or drugs of choice, and major side effects. Selected references are listed at the end of each list. Faculty expand on these key drugs in lectures and key drugs are emphasized in examinations. These Key Drug Lists allow students, and faculty, to focus their attention on important agents. This has increased student interest and mastery of the material. Based on year‐end surveys, these Key Drugs Lists have been extremely well received by the students whose performance on local pharmacology exams and on national board examinations have improved since their initiation.
Oxidative stress on mouse embryonic stem cell proliferation, viability, pluripotency and senescence
Authors: Guo Yan‐Lin , Rajan Suja , Chakraborty Samujjwal
Published: June 2021

Normal cellular activities require a balanced redox environment. Disturbances in this environment may result in oxidative stress, which affects many cellular activities and is considered to be a prominent cause of aging and age‐related diseases. Most of our knowledge in this field is from fully differentiated cells. Embryonic stem cells (ESCs) have attracted enormous attention for their potential applications in cell therapy due to their ability to differentiate to various types of cells and their unlimited capacity for self‐renewal. ESCs are constantly exposed to potential damage by oxidative stress associated with the long‐term in vitro differentiation process and maintenance, but little is known about how they respond to oxidative stress. In this study, we investigated the effects of oxidative stress induced by H2O2 on mouse ESCs and ESC‐differentiated cells. Our results indicated that H2O2 at the concentrations from 10 nM ‐200 µM can inhibit cell proliferation and cause apoptosis depending on the concentration and the duration of the treatment. However, the pluripotency of the ESCs were not significantly affected. Our data suggested ESCs were resistant to the oxidative stress that induced senescence of fibroblasts, however, ESCs‐differentiated cells became susceptible. Our results suggested that ESCs may have superior capacity against oxidative stress than differentiated cells.
Ethanol Enhances Osteogenic Differentiation of Human Amniotic Fluid‐derived Stem Cells
Authors: Hipp Jennifer , Hipp Jason , Atala Anthony , Soker Shay
Published: June 2021

Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental defects caused by prenatal alcohol exposure. Clinical manifestations of FASD are highly variable and include physical, mental and growth retardation, and multiple organ defects. The complex and diverse response of fetal cells to ethanol has prompted us to use a bioinformatics approach to study the effect of ethanol on fetal stem cells derived from the amniotic fluid (AFSCs). Large‐scale transcriptome analysis of ethanol‐treated AFSC showed pathways involved in skeletal development and ossification were up‐regulated, suggesting a basis for ethanol‐induced skeletal malformations in vivo. We therefore examined ethanol's effect on osteogenic differentiation of AFSC. Ethanol treatment during the first 48 hours of osteogenic differentiation increased calcium deposition, and increased both gene and protein expression of alkaline phosphatase. In contrast, ethanol treatment at a midpoint of differentiation (day 8) had no significant effect on the protein expression of alkaline phosphatase. These results suggest that transient exposure of AFSC to ethanol during early differentiation has long‐term effects and enhances osteogenic diffferentiation as indicated by an increase in bone‐specific markers and calcium deposition.
Identification of regenerating myofibers within an extracellular matrix seeded with bone marrow derived cells
Authors: Tierney Matthew , Merritt Edward , Sarathy Apurva , Walters Thomas , Farrar Roger
Published: June 2021

Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials may be capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation of the damaged area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1 x 1 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle‐derived extracellular matrix (ECM). Bone marrow‐derived cells (BMCs) were injected within the ECM 7 days post‐injury. Immunohistochemical identification of desmin, a structural muscle protein, and myogenin, a late transcription factor required for myoblast differentiation, was performed within the defected area. Regenerating myofibers as identified by these markers were visually identified within the ECM following 28 & 42 days recovery. These results demonstrate the ability of ECM to support cellular ingrowth in this model. This work was funded in part by the US Army MRMC Grant DAMD W81XWH‐06‐1‐0540.
The development of biomaterials from porcine skeletal muscle extracellular matrix
Authors: Stern Matthew Michael , Fearing Bailey V. , Soker Shay , Kritchevsky Stephen B. , Christ George J. , Van Dyke Mark
Published: June 2021

There is currently great interest in the development of biomaterials for use in the engineering and/or regeneration of skeletal muscle tissue. Here, we describe the development of an acellular scaffolding system from porcine skeletal muscle extracellular matrix (ECM) molecules. Acellular scaffolds were prepared through the decellularization, lyophilization, and sterilization of thin slices of porcine loin muscle tissue. Myogenic cells seeded onto these scaffolds exhibited the ability to fully penetrate and recellularize the scaffolds. This represents a significant advance over scaffolding systems in which seeded cells fail to penetrate and simply pile onto the scaffold's surface. We also demonstrate that decellularized skeletal muscle tissue can be processed into bioactive powder or gel forms that promote the growth of myogenic cells. Gels composed of skeletal muscle ECM embedded with myogenic cells supported cell growth and gel remodeling. Myogenic cells cultured on surfaces coated with skeletal muscle ECM extract underwent enhanced proliferation relative to uncoated surfaces and simple collagen I coated surfaces. These results demonstrate the feasibility of using bioactive materials comprised of porcine skeletal muscle ECM molecules to produce different classes of biomaterials for use in tissue engineering and regenerative medicine applications.
The effects of Jatropha curcas latex on activation and phagocytotic binding of Saccharomyces cerevisiae in RAW 264.7 macrophages
Authors: Mack Nzinga , Soliman Karam F
Published: June 2021

The latex derived from the subtropical and tropical plant Jatropha curcas (Euphorbiaceae) is commonly used throughout the world for the treatment of dermal abrasions. There is meager data as to the cellular mechanism of action responsible for the observed latex wound healing properties. In this study, we examined the effects of latex on activation of macrophages and the subsequent binding / recognition of Saccharomyces cerevisiae as a model to ascertain potentiating effects for macrophages to bind invasive pathogens. In this study, non‐viable yeasts destroyed by heat and peroxide (3.5x107/ml) were incubated with varying doses of latex in the presence of macrophages. The data indicate that macrophages incubated with latex (1.2 and 1.5mg/ml) for 15 minutes resulted in greater surface binding of yeast (186% and 191%) relative to untreated controls. The data also show that macrophages were not activated by the presence of latex, as assessed by quantification of inducible nitric oxide synthase and nitric oxide after 24 H incubation. In summary, the preliminary findings suggest that the effects of latex may involve greater trafficking of potential pathogens to immune cells, rather than stimulating the expression of pro‐inflammatory proteins. (Supported by a grant from the National Institutes of Health (NIH)/National Centers for Research Resources (NCRR: RR 03020)
Identification of differentially expressed genes in early inner ear development
Authors: Paxton Christian Nephi , Chapman Susan C. , Schoenwolf Gary C.
Published: June 2021

To understand the etiology of congenital hearing loss, we must gain a comprehension understanding of the molecular genetic mechanisms underlying normal ear development. Our lab is identifying genes and their mechanisms of action that lead to inner ear initiation and development. Using Agilent microarrays, we compared the differential expression of the pre‐otic placodal domain with an adjacent rostral control tissue (a domain that is competent, but not specified, to form otic tissue). We identified 1,261 transcripts differentially expressed between the two tissues at a change of 2‐fold or greater: 463 were up regulated in the pre‐otic domain, and 798 were upregulated in the control tissue. We have validated the pre‐otic expression of several signaling molecules and transcription factors identified in this array using in situ hybridization. Furthermore, the expression patterns of this group of genes were explored in detail to determine how the timing of their expression relates to specific events of otic induction and development. In conclusion, a number of novel candidate genes for otic placode induction have been identified. Funded by the Deafness Research Foundation and the NIH.

Grant Funding Source

Deafness Research Foundation and NIH
Hypoxia as an Environmental Cause of Holoprosencephaly
Authors: Smith Francis Joel , Marcucio Ralph S
Published: June 2021

Holoprosencephaly is a complex congenital malformation of the forebrain and face. Recent clinical evidence shows a correlation between hypoxia and holoprosencephaly. We examined the role of hypoxia in causing holoprosencephaly in chick embryos. Chicken eggs were incubated in 13% O2 (hypoxia) or in 20% O2 (normoxic control). Embryos were collected on days 4, 5, 6, and 10. Embryos were photographed for morphologic analysis, sectioned in paraffin, and H&E stained for histologic analysis. 56% of hypoxic embryos (days 4‐6) survived, and 50% survived to day 10. These embryos were mildly or severely developmentally delayed. Malformations included severe cephalic, facial and eye anomalies, and absence of the upper jaw anlage. Another group of eggs were incubated in normoxia to day 7 and transferred to hypoxia. These embryos were collected on day 12 and photographed for morphologic analysis. All the postnormoxic hypoxic embryos (day 12) survived and showed a wide range of stages, compared to normal 12‐day‐old embryos. Malformations included midfacial hypoplasia, midline clefting, and eye anomalies. Hypoxia leads to high mortality, developmental delay, and craniofacial and cephalic malformations resembling holoprosencephaly in chick embryos. Future work will be done to examine the molecular mechanisms of these defects in hypoxia.

Grant Funding Source

NIH
Teaching the History of Anatomy
Authors: Jackowe David J.
Published: June 2021

"Vesalius, who?" Often, medical/graduate students leave their anatomy curricula well trained in the sciences, but with little if any appreciation for the history of the discipline. As such, the history of anatomy is frequently regarded as "emeritus" territory. Here, the author presents a unique curriculum for incorporating the History of Anatomy into a Medical School and/or graduate school course.
Histology lab based on virtual microscopy
Authors: Spornitz Udo Meinhard
Published: June 2021

The shortage of histology slides on the one hand, the omnipresence of personal computers on the other hand, , make virtual microscopy almost a perfect replacement for the old fashioned histology lab. It is no longer to be rated as nice to have, but rather as need to have. Based on the award‐winning vMic (virtual microscopy) developed at the University of Basel (D. Glatz + K. Glatz‐Krieger) which enables students to explore interactively light microscopic slides, the scans of which have been stored on a internet server, virtual microscopy can be carried out with a simple internet browser from any internet access point, 24 h a day. Only one perfect histology slide which has been scanned on an automated microscope for acquisition of a matrix of images must be available to be utilized by hundreds of students. For the acquisition of scans a Zeiss Apochromat (40 x) with numerical aperture (NA) 0.95 was used to obtain high quality images. The images were post processed and then served by a plain http‐server. For the individual histology slide up to several thousand individual scans are used. However, a series of only 4 frames is transmitted to the internet user at the same time, thus the time for downloading is reduced to seconds or fractions thereof. The whole histology slide can be viewed on the browser with magnifications between 1.25x and 40x. Instruction leaflets stored as pdf‐format describe the aim of the individual preparation, the important details, stain, and show also some micrographs marked with arrows and labeled to alleviate the inspection of the slides. A number of multiple choice questions at the end of every instruction leaflet help the students to prepare for the exam.
Cadaver Computed Tomography: Virtual dissection as a tool for teaching anatomy
Authors: Ellozy Sharif H , Lerner Susan M , Reidenberg Joy S , Weintraub Joshua L , Lefkovitz Zvi , Marin Michael L , Laitman Jeffrey T
Published: June 2021

Traditionally, cadaver dissection has been the mainstay of anatomy instruction. However, as clinical medicine becomes more image driven, familiarity with cross‐sectional anatomy is crucial. Additionally, introducing clinical correlates in the preclinical years of medical education is essential in generating student enthusiasm. In order to facilitate these goals high resolution computed tomography was performed on all cadavers used in the first year anatomy class. Axial, sagittal, coronal and 3D volume rendered reconstructions were performed. Specific reconstructions of bony structures, aerodigestive tract, and vascular system were also done. All of the CT scans were read by a radiologist and findings recorded. Students were then given access to all of the studies and instructed on how to use a DICOM viewer. Images from reconstructions were included on their examinations, and part of the students' final grade came from a team building exercise using the CT images. The applicability of this tool to clinical medicine is twofold: 1) it introduces students to cross sectional imaging; and 2) it allows students to identify variant anatomy, post‐surgical changes, and pathology prior to dissection. Unlike other anatomy correlates such as the Visible Human, these reconstructions are of the students' own cadaver, and provide a permanent resource to the student long after the dissection is completed.

Grant Funding Source

n/a
Fail‐safe Termination Elements: A Common Feature of the Eukaryotic Genome?
Authors: Nazar Ross N. , Nabavi Sadegh
Published: June 2021

A recent scan of the human genome identified approximately 11 million hairpins. Some have been linked to known sequences such as viruses, transposable elements and, more recently, regulating or microRNAs but the significance, if any, of most sequences which can be predicted to form hairpins remains unknown. Here we show that hairpins which are cleaved by RNase III‐like nucleases can induce termination, even with normally polyadenylated transcripts and that a cleaved hairpin, downstream of a normal termination signal can induce fail‐safe termination. The results raise the possibility that similar fail‐safe termination elements are widely distributed in the eukaryotic genome to prevent read‐through transcription from disrupting downstream promoter elements. Supported by the Natural Sciences and Engineering Research Council of Canada.
The Effect of the GATA‐1 Transcription Factor in Erythropoiesis.
Authors: White Zach , Damon Christian , Pikaart Michael J
Published: June 2021

GATA‐1 is a key transcription factor that regulates erythrocyte development and maturation. Studies of lead and GATA‐1 have shown that lead decreases the binding affinity of GATA‐1 to DNA. GATA‐1 contains two zinc binding fingers, one at the N‐terminus and one at the C‐terminus, both of which allow tight GATA protein binding to DNA. In purified GATA protein, lead disrupts a GATA‐1 zinc binding finger by replacing the zinc ion with a lead ion, therefore disrupting the protein's conformation and lowering DNA binding affinity. Our current work addresses the implication of lead interfering with GATA function in living cells. Three different cell culture experiments were conducted to test the effects of lead and DNA binding affinity. First, mouse erythroleukemia (MEL) cells were induced by dimethyl sulfoxide (DMSO) with varying lead concentrations and grown for five days to test levels of hemoglobin expression. Second, quail fibroblast (QT6) cells were effectively transfected with GATA plasmids using cationic lipids with different concentrations of lead to determine GATA expression. Third, pluripotent mouse bone marrow cells were cultured for 48 hours to determine erythroid colony formation. These experiments have shown that the addition of lead inhibits hemoglobin production and GATA‐1 expression and that the GATA zinc fingers may be the location where lead binds and inhibits erythropoiesis.
Transcriptional Regulation of N‐Acetyl Glutamate Synthase
Authors: Kirsch Sandra Beth , Marino‐Ramirez Leonardo , Tuchman Mendel , Caldovic Ljubica
Published: June 2021

The urea cycle converts ammonia, a toxic product of protein metabolism, to urea in mammalian livers. N‐acetyl glutamate synthetase (NAGS) catalyzes the formation of N‐acetyl glutamate, an essential cofactor of carbamylphosphate synthetase I (CPSI). By providing an essential activator of CPSI, the first and rate‐limiting enzyme in the urea cycle, NAGS helps regulate urea production and may function as a nitrogen load sensor.

Methods to identify transcription start sites, conserved regulatory regions, and cis‐acting regulatory motifs for NAGS included 5' RACE, multiple sequence alignment of several mammalian species upstream non‐coding sequences, Cis‐element OVERrepresentation software & Chromatin Immunoprecipitation (ChIP). Regions of high conservation were identified immediately upstream and 3kb upstream of the translation start codon. Several transcription factor binding motifs, including Sp1, AR & CREB, were conserved within the putative promoter while a TATA box motif was absent. ChIP analysis verified binding of Sp1 to the NAGS promoter. These data indicate NAGS transcription is regulated through Sp1 and hormone signalling pathways including glucocorticoids and cAMP, similar to regulation of urea cycle enzymes ASS, ASL & arginase I. These finding suggest regulation of NAGS occurs through metabolite sensing pathways and that NAGS could be the first nitrogen load sensor.

NIH # 2R01DK064913‐05A1
Is Dominant Intermediate ‐ Charcot‐Marie‐Tooth type C disorder due to a defect in protein synthesis?
Authors: Froelich Clifford Anthony , Chaudoir Marc W. , Andrews Tara , First Eric A.
Published: June 2021

Charcot‐Marie‐Tooth disorder is the most common inherited peripheral neuropathy, afflicting one in every 2500 Americans. Jordanova et al. have shown that mutation of the tyrosyl‐tRNA synthetase gene is responsible for dominant intermediate Charcot Marie Tooth disorder type C (DI‐CMTC). Three changes in tyrosyl‐tRNA synthetase were found to give rise to DI‐CMTC: replacement of G41 by arginine (G41R), replacement of E196 by lysine (E196K), and deletion of amino acids 153‐156 (Del(153‐156)). To test the hypothesis that DI‐CMTC is due to a defect in the catalytic activity of tyrosyl‐tRNA synthetase, pre‐steady state kinetic methods were used to characterize these three variants. Two of the variants, G41R and Del(153‐156), have severe catalytic defects in their ability to activate tyrosine. The third variant (E196K) activates tyrosine and transfers the tyrosyl moiety to tRNATyr with kinetics that are similar to those of the wild type enzyme. This indicates that a defect in the catalytic activity of tyrosyl‐tRNA synthetase is not responsible for DI‐CMTC. In addition, the stabilities of the DI‐CMTC variants did not differ significantly from that of the wild type enzyme. These observations suggest that DI‐CMTC is not due to a defect in protein synthesis, raising the possibility that it results from a non‐cognate function of tyrosyl‐tRNA synthetase. This research is supported by NIH GM 68070 and MDA 4268 to EAF.
A study of the product profile and substrate specificity of prostaglandin endoperoxide H synthases
Authors: Song Inseok
Published: June 2021

Prostaglandin endoperxide H synthase (PGHS), which catalyzes the first and committed step in prostaglandin synthesis, exhibits both a cyclooxygenase and a peroxidase activity. Initially two molecules of O2 are incorporated into arachidonic acid to form the hydroperoxy endoperoxide PGG2, which is reduced to form the hydroxyl endoperoxide PGH2. There are two isozymes (PGHS‐1 and ‐2), encoded by different genes. Both are structurally and catalytically similar, but show different expression pattern. PGHS‐1 is constitutively expressed, but PGHS‐2 expression is transient and inducible in response to several mitogenic and pro‐inflammatory stimuli. Besides their different expression mode, coupling of each isozyme with the downstream prostanoid synthases is distinctive. For instance, thromboxane A2 is synthesized by functional coupling between PGHS‐1 and thromboxane synthase in platelet cells, which do not express PGHS‐2. Depending on the efficiency of their coupling, not only the designated prostaglandin is formed, but a combination of other prostaglandins can be produced. The product profile was shown to be even more complicated in the presence of reduced glutathione at physiological concentrations, which altered drastically arachidonic acid metabolism. Secondly, the peroxidase active site of PGHS, which is located across from the cyclooxygenase site and hydrophobic dome‐shaped, was investigated to dissect the essential residues for PGG2 specificity.
Catalysis of ester hydrolysis by Escherichia coli carboxyltransferase
Authors: Thalji Nabil , Waldrop Grover L
Published: June 2021

Acetyl‐CoA carboxylase (ACCase) catalyzes the biotin‐dependent carboxylation of acetyl‐CoA to malonyl‐CoA in the first step of fatty acid synthesis in all organisms. Bacterial ACCase consists of two catalytic components: biotin carboxylase (BC), which catalyzes the carboxylation of biotin, and carboxyltransferase (CT) which transfers CO2 from carboxybiotin to acetyl‐CoA to make malonyl‐CoA. The binding sites in CT for carboxybiotin and acetyl‐CoA possess oxyanion holes that are thought to stabilize oxyanionic intermediates during the transfer. We investigated whether these oxyanion holes catalyze ester hydrolysis, which also proceeds through an oxyanionic transition state, using p‐nitrophenyl acetate (PNPA) as an alternative substrate for E. coli CT. By measuring production of p‐nitrophenol (PNP) spectrophotometrically it was found that CT catalyzes PNPA hydrolysis at pH 7.0. Biotin inhibited hydrolysis of PNPA, while malonyl‐CoA increased the rate of hydrolysis. Mutation of an oxyanion hole residue in the acetyl‐CoA binding site did not affect the rate of catalysis. Thus, PNPA hydrolysis likely occurs in the biotin binding site, and binding of acetyl‐CoA enhances binding of PNPA to the enzyme.
Evaluation of docosahexaenoic and arachidonic acid as modifiers of carnitine palmitoyltransferase in pre‐ and term pigs
Authors: Martin Jessica E , Campbell Jenny A , Brockson M. Elizabeth , Melendez Krizia , Lyvers Peffer Pasha A
Published: June 2021

Pigs were delivered by cesarean at 106 or 114d of gestation and fitted with umbilical catheters. Pigs received either a control, a low DHA/AA (0.3 and 0.6% of total lipids as DHA and AA, respectively), or a high DHA/AA (5% and 11 % of total lipids as DHA and AA, respectively) total parenteral diet for 6d. Hepatic CPT activity was measured before and after malonyl‐CoA inhibition. There was a tendency for increased total CPT activity in preterm compared to term pigs receiving the control diet (p = 0.07) and was attributed to malonyl‐CoA insensitive activity, which was elevated across dietary treatments in preterm versus term pigs (p<0.05). This increased CPT activity was concomitant with increased CPT I transcript amounts (p < 0.05). Whereas the low DHA/AA diet resulted in postnatal upregulation of hepatic CPT I mRNA in term pigs, the control and high DHA/AA diet elicited a similar response in CPT I mRNA in preterm pigs (p < 0.05). It is unlikely that the differences observed in CPT can be ascribed to PPARα as transcript amounts were increased in term pigs receiving the control and low DHA/AA diets (p < 0.05), yet postnatal changes in PPARα were not observed in preterm pigs receiving the high DHA/AA diet. Collectively, these findings suggest that postnatal induction of hepatic CPT I is more robust in preterm pigs. This research has been supported by funds appropriated by the Ohio General Assembly to The Ohio State University, Ohio Agricultural Research and Development Center.
Sequence variation in the human dopamine transporter gene in subjects with bipolar disorder
Authors: Miller Leah , Mergy Marc , Mckinney Rebecca , Kelsoe John , Blakely Randy
Published: June 2021

The dopamine (DA) transporter (hDAT, SLC6A3) plays a critical role in terminating synaptic DA neurotransmission and maintaining DA homeostasis. Dysfunction of hDAT is believed to contribute to various neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder (BPD). Recently, we identified a rare hDAT variant, Ala559Val, in two ADHD subjects, which has also been identified in a subject with BPD [Grunhage et al., 2000]. In this study, we are evaluating whether functional variants in hDAT exist in BPD and ascertain the transmission pattern and functional nature of identified polymorphisms. We utilize PCR and temperature‐gradient capillary electrophoresis (TGCE) to screen for mutations in the entire coding region and adjacent non‐coding sequence of the hDAT gene in a cohort of >400 BPD (Type 1 and II) subjects. Preliminary analyses reveal a number of both novel and common single nucleotide polymorphisms (SNPs) across the gene. Our study constitutes the largest analysis to date of DAT coding sequences in BPD, permitting the identification of both common and rare variants that could contribute to DAT dysfunction in the disorder. Supported by NIH grant #T32NS06120106 and T32MH064913; NIMH grant # MH073991, MH47612, MH59567, MH68503 and Veterans Affairs VISN 22 Mental Illness Research, Education, and Clinical Center.
systematic analysis of a simple adaptor protein PDZK1: ligand identification, interaction and functional prediction of complex
Authors: Hu Siqi , Song Eli , Tian Rui , Ma Sucam , Yang Tao , Mu Yi , Li Yuan , Shao Chen , Gao Shijuan , Gao Youhe
Published: June 2021

PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two‐hybrid screening of a random peptide library and high‐throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three‐hybrid system. Many G‐protein signaling pathway‐related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide‐binding domains.
Evaluating the Effect of Applying the FDA Definition of Whole Grains to Health Claims for Risk Reduction of Cardiovascular Disease and Diabetes
Authors: DeMoura Fabiana F , Lewis Kara D. , Mares Julie , Sapirstein Harry , Marlett Judith , Hoadley James , Falk Michael C.
Published: June 2021

The U.S. Food and Drug Administration (FDA) defines whole grains as consisting of the intact, ground, cracked or flaked fruit of the grains whose principal components‐the starchy endosperm, germ and bran‐are present in the same relative proportions as they exist in the intact grain. We evaluated the effect of applying the FDA definition of whole grains on the strength of scientific evidence in support of claims for risk reduction of cardiovascular disease (CVD) and diabetes. We concluded that using the FDA definition for whole grains as a selection criterion is limiting because the majority of existing studies often use a broader meaning to define whole grains. When considering only whole grain studies that met the FDA definition we found insufficient scientific evidence to support a claim that whole grain intake reduces the risk of CVD. However, a whole grain and CVD health claim is supported when using a broader concept of whole grain to include studies that included intake of bran and germ as well as whole grain. The scientific evidence on the relationship of whole grain consumption and diabetes is suggestive but inconclusive whether or not the definition of whole grains was in accordance with that of the FDA. This type of analysis is complicated by variation among different types of whole grains due to their diversity in nutrients and bioactive components. This project was sponsored by Kellogg Company, USA.
N‐3 fatty acid treatment and plasma transthyretin in patients with Alzheimer's disease
Authors: Faxen Irving Gerd , Freund‐Levi Yvonne , Eriksdotter‐Jönhagen Maria , Basun Hans , Palmblad Jan , Vedin Inger , Wahlund Lars‐Olof , Cederholm Tommy
Published: June 2021

Background

Transthyretin (TTR) binds Abeta and may reduce brain Abeta, a key pathogenetic feature in Alzheimer's disease (AD). N‐3 fatty acids (FA), i.e. DHA and EPA, increase TTR transcription in rat brain. We studied the effects of n‐3 FAs on plasma TTR levels in patients with AD. Methods: 204 outpatients with AD were randomised to DHA/EPA (80/20%), 4 g/d during 6 mo (EPAX, Pronova AS) or placebo (n‐6 FA). After 6 mo all patients received EPAX for another 6 mo. Cognitive function was assessed by MMSE and ADASCog.

Results

Baseline plasma TTR was 260[plusmn]80 in the EPAX‐group (89 pat, 75 y, 57% w) and 264[plusmn]55 mg/l in the controls (85 pat, 75 y, 46% w) (ns). After 6 mo TTR levels was 250[plusmn]59 and 229[plusmn]51 in the EPAX and control groups, respectively (p<0.015). Repeated measures ANOVA indicated an increase in TTR by EPAX treatment (P=0.04), most evident in the men. From 6 to 12 mo TTR increased significantly in both groups. MMSE correlated to TTR at 12 mo (r=0.16, P=0.03) and inversely to ADAS‐Cog (r=‐0.2, P=0.01). In multiple regression analyses increased TTR at 6 mo was independently related to n‐3 treatment (OR 2.1, 95% CI 1.01‐4.2)

Conclusion

N3 FA treatment in patients with AD appeared to increase TTR. Since n3 FA may prevent cognitive decline, and TTR may decrease Abeta deposition, the results warrant further exploration. & [beta] & [plusmn]
N‐3 polyunsaturated fatty acids do not alter immune and inflammation measures in endurance athletes
Authors: Henson Dru A. , Nieman David C. , McAnulty Steve , Jin Fuxia , Maxwell Kendra
Published: June 2021

We tested the influence of 2.4 g/day n‐3 polyunsaturated fatty acids (n‐3 PUFA) over 6 weeks on exercise performance, inflammation, and immune measures in 23 trained cyclists before and after a 3‐day period of intensified exercise. Subjects were randomized to n‐3 PUFA (N=11) (2,000 mg eicosapentaenoic acid, EPA, 400 mg docosahexaenoic acid, DHA) or placebo (N=12) groups, and ingested supplements under double blinded methods for 6 weeks prior to and during a 3‐d period in which subjects cycled for 3 h/day at ~57% Wattsmax with 10‐km time trials iduring the final 15 minutes of each 3‐h bout. Blood and saliva were collected before and after the 6‐week supplementation period, immediately following the 3‐h exercise bout on the third day, and 14‐h post‐exercise. Supplementation with n‐3 PUFA significantly increased plasma EPA and DHA, but had no effect on 10‐km time trial performance, pre‐exercise measures, and exercise‐induced increases in plasma cytokines, myeloperoxidase, blood total leukocytes, serum C‐reactive protein, and creatine kinase, or the decrease in the salivary IgA:protein ratio. In conclusion, 6 weeks supplementation with n‐3 PUFAs increased plasma EPA and DHA, but had no effect on exercise performance or in countering measures of inflammation and immunity before or after a 3‐day period of nine hours of heavy exertion.

Supported by a grant from Cooper Complete, The Cooper Aerobics Center, Dallas TX
The effect of omega‐3 fatty acids derived from krill protein concentrate on tissue deposition and lipid peroxidation
Authors: Bridges Kayla , Gigliotti Joseph , Jaczynski Jacek , Tou Janet C.
Published: June 2021

Krill protein concentrate (KPC) has been determined to be a high quality protein for human consumption with the advantage of being a rich source of the omega‐3 polyunsaturated fatty acids (ω‐3 PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The ω‐3 PUFAs in krill oil are mainly associated with phospholipids, which have been proposed to result in high incorporation of ω‐3 PUFAs into tissues and to be stable against oxidation. The study objective was to determine tissue deposition and oxidative stability of ω‐3 PUFAs in rats fed KPC. Young female Sprague‐Dawley rats (n=10/group) were fed ad libitum isocaloric diets with either 10% freeze‐dried KPC (~8% oil) containing 4.4% corn oil (KPC + CO) or 10% casein containing 5.3% corn oil (C + CO) for 4 weeks. Fatty acid compositions of various tissues were analyzed by gas chromatography. Lipid peroxidation was determined by TBARS and total antioxidant capacity by enzyme immunoassay. The study results showed even at a KPC contribution of 0.9% lipids to the total diet, EPA and DHA content increased (P<0.01) in adipose and liver tissue while decreasing (P<0.01) the ω‐6 PUFA, arachidonic acid. There was no significant difference in brain tissue deposition of ω‐3 PUFAs, total antioxidant capacity, or lipid peroxidation between groups. Based on the study results, ω‐3 PUFAs associated with KPC resulted in tissue incorporation without changing oxidative stability.
The Effect of Whey Protein on Post‐Meal Blood Glucose and Insulin
Authors: Akhavan Tina , Luhovyy Bohdan L , Anderson Harvey G
Published: June 2021

Dairy protein ingestion is associated with satiety and the reduction of blood glucose (BG) in response to carbohydrate. The objective of this experiment was to examine the effect of whey protein on pre‐ and post‐meal glucose and insulin. Healthy normal weight adult males (n = 10) and females (n = 8) were provided whey protein (5, 10, 20 and 40 g) and hydrolyzed whey protein (10 g) in 300 ml of water or water alone 30 min prior to a pizza meal (12 kcal/kg body weight). Insulin and BG were measured by finger prick at baseline and at intervals pre‐and post‐meal for 170 min. Pre‐ (0‐30 min) and post‐ (30‐170 min) meal areas under the curve (AUCs) for BG and insulin were calculated. Sex (p <0.05) and whey protein (p <0.001) affected BG and insulin responses. Females had higher post‐meal BG and insulin AUCs than males (n =18, p < 0.05). Whey protein (10 g to 40 g) increased pre‐meal insulin and BG but reduced post‐meal BG and insulin AUCs (n = 18, p <0.05). The lower post‐meal BG AUC after whey protein was not associated with a higher insulin AUC compared with the control suggesting that whey protein treatments improved post‐meal insulin sensitivity. We concluded that the ingestion of whey protein prior to a meal resulted in lower post‐meal BG and insulin and reduced the amount of insulin required for the post‐meal glucose response.

Grant Funding Source

Natural Sciences and Engineering Research Council of Canada (NSERC) and Kraft Ins.