bluuebunny/crossref_metadata_2025 huggingface dataset:
Complex curvilinear allometry of brain size scaling in mammals
Authors: Albrecht Gene H. , Gelvin Bruce R. , Miller Joseph M. A.
Published: June 2021

The scaling relationship between log(brain size [E]) and log(body size [P]) is usually described by linear regression using the simple allometry equation log(E) = log(k) + a · log(P). The k ≈ 0.67 of early studies was thought to reflect the geometric scaling of brain volume to body surface area. The k ≈ 0.75 of later work was thought to reflect metabolic rate which also scales to the ¾ power of body size. We use data from the literature on 856 mammalian species to show that such coefficients are artifacts of forcing a linear model on curvilinear data. Brain size scaling is best described by the second‐order polynomial log(E) = log(k) + a1 · log(P) + a2 · [log(P)]2 fit by least‐squares polynomial regression. “Instantaneous allometry coefficients” (ki; i.e., the slope of the tangent to the curved polynomial regression line analogous to the k's of simple allometry) decrease as body size increases (e.g., ki = 0.89 for 10 g species to ki = 0.49 for 10,000 kg species). The same pattern of complex allometry holds for the individual mammalian orders with the notable exception of marsupials whose ki's increase as body size increases. Besides requiring revisions to biological explanations of brain size scaling, our finding of complex curvilinear allometry affects predictions of brain size from body size as well as calculation of encephalization quotients (EQ) used as a comparative measure of intelligence.
Comparative morphology of the rat optic nerve using light and electron microscopy
Authors: Duncan Joshua Kyle , Anderson Bryan , Kaufman Jason A. , Iwaniuk Andrew , Jones T. Bucky , Bray Douglas , Suchocki Lawrence , Hall Margaret I.
Published: June 2021

Optic nerve fibers are the axons of retinal ganglion cells, and as such they can provide information about the visual acuity and sensitivity capabilities of an animal. Consistent optic nerve fiber counts are not currently available for most animals. In this study, using light microscopy, we counted optic nerve fibers for 7 nerves from 4 adult Sprague‐Dawley rats. Using unbiased stereology we counted 4 adjacent sections of each nerve at 1000X, each using a different staining technique, including Bielschowsky's silver impregnation, Bodian's protagarol, Luxol Fast Blue stain, and a combination Bodian's protagarol and Luxol Fast Blue. We obtained counts of 75,000 to 90,000 fibers, with a fiber size range of 0.1μm to 5μm. We also examined preliminary electron micrographs of one Sprague‐Dawley rat optic nerve at magnifications up to 10,000X. Published counts of the pigmented rat optic nerve fibers using electron microscopy are 120,000, with a size range of 0.4μm to 5μm. This is significantly higher than our light microscopy counts. However, using EM at 10,000X we observe myelinated fibers as small as 0.02.μm. This discrepancy in fiber numbers calls into question which methodology is most suitable for counting myelinated fibers within the optic nerve.

Grant Funding Source: Kenneth A. Suarez fellowship
Eye size, flight speed, and Leuckart's Law in birds
Authors: Heesy Christopher Paul , Hall Margaret I.
Published: June 2021

Birds have the largest eyes, both relatively and absolutely, of any of the terrestrial vertebrates. Large avian eye size has been hypothesized to be an adaptation to flight as part of Leuckart's Law, the idea in biology that more swiftly moving animals have larger eyes. Increased spatial resolution is one result of larger eye sizes and may possibly improve an animal's ability to judge distances, of obvious importance for flight. Leuckart's Law in birds has previously been tested utilizing Plasticine eye models and body mass as a surrogate for flight speed. In this study, we test Leuckart's Law using axial length measurements of eyeballs obtained from wet bird specimens and flight speeds obtained from flying birds. We find no evidence to support Leuckart's Law: for 98 bird species across 6 orders, a regression of eye axial length vs. flight speed explains virtually none of the variance, with an r2 value of 0.001. Ducks, the fastest‐flying birds in our sample, exhibit only moderate eye sizes, while falcons, the largest‐eyed birds measured, have only moderate flight speeds. We therefore conclude that Leuckart's Law is not useful for explaining bird eye sizes.
Effects of a high soy protein diet on forelimb function after stroke in rats
Authors: Burnet Derek , Strieker Shanna M. , Butteiger Dustie N. , Banz William J. , Cheatwood Joseph L.
Published: June 2021

Dietary strategies aimed at increasing post‐stroke outcomes are lifestyle alterations which could be easily implemented by people at risk of stroke. Soy diets have been demonstrated to provide some benefits in the short term following stroke, but longer time periods have not been studied. Further, carefully defined diets containing soy protein isolates have not been investigated. In the current study, male LE Hooded rats were fed semi‐ purified diets containing either sodium caseinate or a soy protein isolate. Rats were trained to perform the skilled forelimb reaching task and subsequently underwent unilateral middle cerebral artery occlusion (MCAO) to induce a stroke lesion. After stroke, rats remained on the same diet and were tested daily for a period of 8 weeks to observe their performance on the skilled forelimb reaching task. In the first and second weeks following stroke, rats which were fed the soy protein diet demonstrated less severe reaching deficits than rats fed the casein diet (p<0.05). Further, rats fed the soy protein diet reached statistical recovery to within baseline range at three weeks post‐stroke. Casein‐fed rats did not reach statistical recovery within the time course of the study. These results indicate that a diet rich in soy protein provides some protection from neurological damage following MCAO stroke in rats and may encourage a more rapid spontaneous recovery of skilled forelimb reaching.

Grant Funding Source: none
Sural nerve morphology in hypertensive rats with chronic diabetes
Authors: Sanada Luciana Sayuri , Tavares Marcelo Rodrigo , Neubern Milena , Fazan Valéria Paula
Published: June 2021

Despite that the association between hypertension and diabetes is common in clinical practice, the contribution of each isolated entity in the neuropathy development is still not known. The literature lacks a morphologic description of peripheral nerves in experimental models of hypertensive diabetic animals. The present study compared the sural nerve in Wistar rats (WR), WR with chronically induced diabetes, spontaneously hypertensive rats (SHR) and SHR with chronically induced diabetes, using a morphological approach. Female SHR and WR, 8 weeks old, received a single intravenous injection of streptozotocin (STZ) through the caudal vein. Twelve weeks after the injection, right and left sural nerves were dissected and proximal and distal segments were prepared for light microscopy. Morphometry was performed with the aid of computer software. Fascicular and myelinated fiber data from sural nerves in control and diabetic WR are statistically larger compared to SHR and diabetic SHR. Hypertension did alter the morphology and morphometry of the fascicles and myelinated fibers of the sural nerves in control SHR compared to control WR. Chronic diabetes also induced alterations of the morphology and morphometry of sural nerve. Hypertension alteration caused an exacerbation of the diabetic alterations already observed in neuropathic degeneration of sural nerves.

Grant Funding Source: FAPESP
Morphological analysis of dentate granule neurons in the mouse model of Fragile‐X syndrome
Authors: Nahirney Patrick Charles , Eadie Brennan , Prescott Jason , Christie Brian
Published: June 2021

Fragile X syndrome (FXS) is the most commonly inherited form of mental retardation and is characterized by the absence of the expression of the fragile‐X mental retardation protein (FMRP) from the Fmr1 gene. It is postulated that dendritic spine abnormalities may underlie intellectual impairment in FXS. The current research was conducted to investigate the morphological changes associated with FXS specifically within the dentate gyrus (DG) of the hippocampus. Animals were genotyped and the brains from Fmr1 knockout (KO) mice and wild‐type (WT) littermates were processed for Golgi‐Cox staining and transmission electron microscopy (TEM). Dendrites of dentate granule neurons from the molecular layer were examined and their spines quantified. Golgi‐Cox analysis by light microscopy revealed fewer spines in the DG of the mouse model of FXS. TEM data analysis showed a trend towards fewer spine apparati in dendritic spines and increased thin spines in Fmr1 KO mice. These data indicate that there are fewer dendritic spines which may be less mature in appearance in the DG of the mouse model of FXS, contrasting findings from other regions of the brain (e.g., visual cortex). These results provide new data on the structure‐function relationship between dendritic spine abnormalities and intellectual impairment in FXS.
Renshaw cell loss in a transgenic mouse model of amyotrophic lateral sclerosis
Authors: AlMarsoummi Sarmad , Blanchard Stephane , Kulikov Sergei , Smith Margaret , Carr Patrick
Published: June 2021

The distribution and abundance of immunohistochemically‐identified Renshaw cells was studied in the lumbar spinal cord of an amyotrophic lateral sclerosis (ALS) mouse model. Transgenic G93A‐SOD1 mice displayed ALS‐like disease with the first neurological sign occurring at 90 days of age and progression at 100 to 150 days of age. Gephyrin and calbindin D28k immunofluorescence were used to identify and count the putative Renshaw cells in lumbar spinal cord tissue from mice at 40, 70, 90 and 145 days of age. Preliminary results suggest that in this model of ALS, functional neurological deficits are preceded and accompanied by a profound loss of gephyrin‐ and calbindin D28k labeled putative Renshaw cells. In 30 micron sections of lumbar spinal cord, mean counts per ventral horn were: at 40 days ALS=1.73 and control=3.13; at 70 days ALS=2.00 and control=3.32; and at 90 days ALS=2.22 and control=3.28; and at 145 days ALS=1.39 and control=3.08. Differences were significant (p<0.05; Mann‐Whitney Rank Sum test) for all ages. A significant decrease in number of Renshaw cells weeks prior to the onset of the disease suggests that, in the B6.Cg‐Tg(SOD1‐G93A)1Gur/J transgenic mouse model, the loss of motor‐related interneurons precedes clinical neurological deficit onset and progression of the disease and the loss of Renshaw interneurons occurs before the onset of motor neuron death.

Supported by NIH 5R01HL075034‐05.

Grant Funding Source: NIH
On the developmental origin of hepatic sinusoidal endothelium: confirmation of angiocirculatory vasculogenesis
Authors: Meyer Anna C. , Sherer Glenn K.
Published: June 2021

Developmental mechanisms reported to account for the origin of hepatic sinusoidal endothelium include (1) in situ differentiation of mesenchymal cells (Sherer, 1991), (2) sprouting from preexisting vessels (Gouysse et al., 2002), (3) transdifferentiation of mesothelial cells (Pérez‐Pomares et al., 2004), and (4) recruitment of angioblasts and/or vascular endothelial cells (VECs) from the circulation (Pardanaud & Eichmann, 2006; Zhang et al., 2006). The present study was designed to investigate whether the last of these mechanisms, here termed angiocirculatory vasculogenesis, plays a role in the development of hepatic sinusoidal endothelium. Chick livers were harvested from quail/chick parabioses maintained either in ovo or in shell‐less culture and were examined for evidence of quail VECs by immunostaining with QH1 antibody. Numerous positively‐stained cells were found to contribute to the sinusoids of parabiotic chick livers. Non‐parabiotic chick liver, serving as a negative control, lacked staining. These findings confirm that at least some of the cells that form these capillary‐level vascular structures are, as previously reported, acquired from circulating blood.

Grant Funding Source: None
Pretreament with losartan affects the inflammation following cardiac injury
Authors: Le Saux Claude Jourdan , Miyasato Shelley , Rueli Rachel
Published: June 2021

Activation of the local renin‐angiotensin system (RAS) following cardiac injury participates in remodeling process. We hypothesized that mice treated with losartan, an angiotensin II receptor blocker, will prevent TGF‐b signaling activation leading to attenuated remodeling.

C57BL/6J mice, 8–10 weeks old underwent cryoablasion of the left ventricle. At day 21 mice received losartan (0.6g/l) in their drinking water until sacrificed. Heart tissue was collected at several time points. The inflammatory response was determined by characterization of immune cell infiltration and cytokine mRNA abundance. Cardiac remodeling was assessed by extracellular matrix gene expression and quantification of collagen deposition by picrosirius red stain. TGF‐b signaling activation was determined by western blot analysis.

All mice showed a peak of inflammation at day 3, and resolution by day 14. Pretreatment with losartan significantly increased macrophage infiltration. It significantly increased TNF‐a; mRNA at days 3 and 14. The level of TGF‐b mRNA was not affected by losartan, and none of the TGF‐b signaling pathways were activated. Correspondingly, collagen deposition did not significantly change.

In contrast to other studies where losartan was given at the onset of injury, treatment prior to induction of cardiac injury may enhance inflammation, while modestly attenuating fibrosis.

Grant Funding Source: NIH‐NCRR RR016453, Hawaii Community Foundation 20080485
Aging reduces differentiation‐associated gene expression in cardiac side population cells that is not attenuated by caloric restriction
Authors: Mulligan Jake , Schmuck Eric G. , Ertel Rebecca L. , Bauwens Jake D. , Saupe Kurt W.
Published: June 2021

The aged heart displays a loss of cardiomyocyte number and function, possibly due to decreased regenerative potential of cardiac progenitor cells. An important cardiac progenitor population that has not been studied in the context of aging is cardiac side population cells (CSPC). To address this, flow cytometry‐assisted cell sorting was used to isolate CSPC from adult (~5‐months old) and aged (~25‐months old) C57Bl/6 mice that were fed either a control diet or an anti‐aging (caloric restriction, CR) diet. Aging caused a 2.0‐fold increase in both the total number of CSPC and in the highly cardiomyogenic sca1+/CD31‐ subpopulation (n=8–10 mice/group). Telomerase activity (a marker of proliferation) in CSPC was not altered by aging (n=4 mice/group). In contrast, aged CSPC showed reduced expression of several genes associated with differentiation, including smooth muscle actin, cardiac muscle actin, and bone collagen type I (5.1‐, 3.2‐, and 2.2‐fold respectively; n=4/group). These data suggest that any loss of regenerative potential in aged CSPC is likely due to inefficiency of differentiation into cardiac lineages rather than poor proliferation rate. None of these age effects were altered by CR diet. Therefore it appears that the manner in which CSPC (and perhaps other types of progenitor cells) age is distinct from the post‐mitotic tissue aging that can often be attenuated by CR.
The Role of Organelle‐Targeted DNA Polymerases in Mitochondrial DNA Replication and Recombination in A. thaliana
Authors: Brammer Jeff , Nielsen Brent L
Published: June 2021

Two putative organelle‐targeted DNA polymerase genes have been identified in the nuclear genome of Arabidopsis thaliana. It has been determined that both polymerases are dual targeted to the mitochondria and chloroplast. In order to better understand the roles these polymerases have in organellar DNA replication and to determine if they have overlapping functions, we have produced crosses that will knockout or knock down expression of these polymerases. We started with plants which had T‐DNA inserts in each individual DNA polymerase gene. By crossing these plants, progeny were obtained that were heterozygous for one T‐DNA insert in both genes. Mutants with inserts in only one of the genes lack any noticeable phenotypic differences. These plants were then crossed to produce plants with various numbers of inserts in both polymerase genes. These plants will be examined for effects on growth, and mRNA levels will be measured for each polymerase in plant samples with homozygous inserts in the polymerase genes as compared to wild type and heterozygous plants. These plants will also be used to compare mtDNA copy number between samples to see if knockdown of these genes causes a reduction in replication.
Laboratory Evolution of Laccase for Substrate Specificity
Authors: Gupta Nirupama , Lee Frederick S , Farinas Edgardo T
Published: June 2021

A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild‐type CotA oxidizes ABTS (ABTS = diammonium 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonate) and SGZ (SGZ = 4‐hydroxy‐3,5‐ dimethoxy‐benzaldehyde azine), and it was engineered for increased specificity for ABTS. Laccases have several applications in biotechnology that include pulp bleaching, biosensors, bioremediation, and biofuel cells. Based on the ABTS‐bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half‐life for the heat inactivation (t1/2) at 80 oC was increased by 62 min for the mutant, and this was surprising since this property was not included in the substrate specificity screen. In short, the substrate‐binding pocket of CotA is moldable and it can be tailored to oxidize a variety of target molecules for specific purposes.
The structure of the mammalian RNase H2 complex provides insight into RNA:DNA hybrid processing to prevent immune dysfunction
Authors: Shaban Nadine Musa , Harvey Scott , Perrino Fred W. , Hollis Thomas
Published: June 2021

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA:DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi‐Goutieres syndrome (AGS). We have determined the crystal structure of the three protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein‐protein interaction motifs that could allow for efficient participation in multiple cellular functions. Further structural and biochemical analyses show that some RNase H2 disease‐causing mutations likely result in aberrant protein‐protein interactions while the RNase H2A subunit‐G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification. Funding: NIH‐R01‐GM069962, Alliance for Lupus Research, ACS.
A role of microRNA in the regulation of hTERT expression?
Authors: Yeh Yuan‐Ming , Huang Pei‐Rong , Wang Tzu‐Chien
Published: June 2021

Micro (mi)RNAs are small noncoding RNAs that control gene expression post‐transcriptionally either via the degradation of target mRNAs or the inhibition of protein translation. Each miRNA is believed to regulate the expression of multiple mRNA targets and many miRNAs have been linked to tumorgenesis, differentiation, immune system and cell death. Since the telomerase reverse transcriptase (hTERT) is an important gene involved in the development of cancers, we investigated whether or not hTERT may be regulated by miRNAs. A search for the presence of targeting sites in the 3’UTR of hTERT mRNA by TargetScan and Miranda algorithm has uncovered a number of miRNAs may contain putative binding sequences. Among them, seven miRNAs (miR‐138, miR‐181c1, miR‐363, miR‐512‐5p, miR‐548a‐3p, miR‐632, and miR‐648) that contain two or more than two putative binding sites were examined in this study to evaluate their effect on the translation of a luciferase reporter which contain the 3’UTR sequence of hTERT mRNA at the 3’ end of luciferase gene. Our preliminary results indicated that none of the 7 miRNAs tested had any significant effect on the expression of luciferase, suggesting that the 3’UTR of hTERT mRNA may not be the target of regulation by miRNAs. This study was supported by research grants EMRPD 180091 and CMRPD 170541.
Towards a chemical anatomy: new tools to image biological molecules in cells and in tissues
Authors: Salic Adrian
Published: June 2021

Microscopic imaging has revolutionized our understanding of the structure and function of cells and tissues. At its best, microscopy can detect cellular components with high sensitivity and spatial resolution. Additionally, live imaging allows dynamic cellular processes to be studied in a non‐invasive manner, with high temporal resolution.

In the last decade, a small number of chemical reactions have been developed which occur with high efficiency and specificity under mild conditions, in aqueous media. These reactions are bio‐orthogonal, involving chemical groups not found in cells and inert toward naturally occurring chemical groups. These reactions are thus ideal for labeling and detecting molecules in vivo. One focus of our lab is to develop chemical technologies for metabolic labeling and direct imaging of biological molecules, based on these bio‐orthogonal chemical reactions. My talk will present our recent work on devising novel biosynthetic probes to image nucleic acids and lipids in cells and in tissues.
Domain II of the HCV IRES is a potent activator of PKR
Authors: Toroney Rebecca , Nallagatla Subba Rao , Cameron Craig E , Bevilacqua Philip C
Published: June 2021

The protein kinase PKR is an essential component of the human innate immune response. In the presence of certain RNAs, PKR is autophosphorylated, which enables it to phosphorylate its substrate, eIF2α, leading to shut down of cellular translation. Activators of PKR are typically long double‐stranded RNAs (≥33 bp) present during viral infection. Recent studies have indicated that the internal ribosome entry site (IRES) in the 5’‐UTR of hepatitis C virus (HCV) RNA can also activate PKR1. The HCV IRES has a complex secondary structure consisting of four distinct domains. While it has been shown that domains III and IV of the IRES activate1, we report that Domain II can also potently activate PKR independent of the other IRES domains. We present analysis of the structural elements of this domain and find that while the double‐stranded helical regions of the RNA are important for activation, the loop regions of Domain II are also important. PKR activation by Domain II of the IRES alone has implications for the cell's immune response when the other domains of the IRES may be inaccessible during various stages of the HCV life cycle. Supported by NIH GM 58709
The effect of bacterial challenge on ferritin regulation in Aedes aegypti
Authors: Geiser Dawn L. , Zhou Guoli , Kohlhepp Edwin A. , Mayo Jonathan J. , Winzerling Joy J.
Published: June 2021

In mammals, ferritin is a cytoplasmic iron storage protein made up of 24 heavy and light chain subunits that can store up to 4500 iron atoms; as wells as an acute phase protein that is secreted upon inflammation. Here we characterize the message and protein expression profiles of Aedes aegypti (yellow fever mosquito) ferritin heavy chain (HCH) and light chain homologue (LCH) subunits in response to iron and bacterial challenge. Mosquito experiments show an increase in midgut HCH and LCH message and protein expression 24 h post‐blood meal (PBM) and decrease 72 h PBM. Fat body and ovary HCH and LCH message and protein expression showed a time‐dependent increase PBM. Heat‐killed bacteria (HKB) had little effect on mosquito tissue ferritin expression. Iron regulatory protein (IRP) binding activity for the iron responsive element (IRE) increased over time in ovary PBM. Midgut and fat body binding activity increased 24 h PBM with a decrease 72 h PBM. Mosquitoes treated with HKB showed no change in IRP/IRE binding activity compared to naïve animals. Cell studies support in vivo results. HCH and LCH message and protein expression reveal a time‐dependent, synergistic up‐regulation of expression in CCL‐125 cells treated with iron and HKB than iron alone. There was also a time‐dependent increase in secreted ferritin as well as decrease IRP/IRE binding activity in cells treated with iron and HKB. Research support from NIH (GM056812), the Agricultural Experiment Station and CALS at the University of Arizona.
[1, 2‐13C]‐Acetate metabolism in glutamate dehydrogenase mutants of Saccharomyces cerevisiae
Authors: Trotter Pamela J , Tang Yijin
Published: June 2021

Glutamate dehydrogenases (GDH) interconvert 2‐oxoglutarate and glutamate. In yeast, NADP‐dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from 2‐oxoglutarate, while an NAD‐dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in nonfermentable acetate/raffinose (Ace/Raf) to examine the role(s) of these enzymes during aerobic metabolism. The doubling time of wt, gdh2Δ, and gdh3Δ cells was comparable at ~ 4 hours. NADP‐dependent GDH activity (Gdh1p + Gdh3p) in wt, gdh2Δ, and gdh3Δ was decreased ~80% and NAD‐dependent activity (Gdh2p) in wt and gdh3Δ was increased ~20‐fold in Ace/Raf as compared to glucose. Cells carrying the gdh1Δ allele did not grow in Ace/Raf, yet both the NADP‐dependent (Gdh3p) and NAD‐dependent (Gdh2p) GDH activity was ~3‐fold higher. Metabolism of [1, 2‐13C]‐acetate and analysis of carbon NMR spectra was used to examine glutamate metabolism. Analysis of 13C‐isotopmers and enrichment of glutamate carbons indicate a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wt. Enrichment of 13C of glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at < 15% of wt. These data suggest that Gdh1p is the primary GDH enzyme, even under nonfermentative conditions, and Gdh2p and Gdh3p play a noticeable role in glutamate metabolism. (Supported by NIH grant GM069372 [P. J. T.])
O‐GlcNacylation of Glycogenin Inhibits Glycogen Synthesis
Authors: Viep Tien , Chima‐Okerke Onyinyechi , Le Pauline , Khatra Balwant
Published: June 2021

It is becoming evident that increased cellular glucose concentration leads to an increase in UDP‐ß‐N‐Acetylglucosamine (UDP‐GlcNac) concentration and O‐GlcNacylation of many proteins. Since glycogenin self‐glycosylates with UDP‐glucose, we investigated whether UDP‐GlcNac serves as its substrate and possible effects of this modification on glycogen synthesis. Incubation of purified glycogen synthase, glycogenin, and apo‐glycogenin with UDPGlcNac led to incorporation of ~0.5 mole of GlcNac per mole of glycogenin. The presence of incorporated GlcNac into glycogenin was confirmed by HPAEC‐PAD and by radiolabeling with galactosyl transferase. The apparent KM for UDP‐GlcNac was 227 μM. Mutating Tyrosine 194 to Phenylalanine eliminated the ability of glycogenin to autoglycosylate with either substrate confirming that GlcNac was added to the same site as glucose. Our results suggest that GlcNac is added to Tyr194 either as a first residue or to the growing oligosaccharide. Furthermore, we observed that recombinant glycogenin modified with O‐GlcNac was still able to autoglucosylate. However, O‐GlcNac modified glycogenin was resistant to the addition of glucose by glycogen synthase. These results suggest that O‐GlcNacylation of glycogenin may inhibit glycogen synthase activity resulting in inhibition of glycogen synthesis. Supported by sabbatical leave to BSK and RISE grant: GM‐071638.
Markers of Insulin Resistance during HVC Treatment: A Relationship to Sustained Virologic Response
Authors: Desai Darshan Veljibhai , Estep Michael , Afendy Arian , Hossain Noreen , Biredinc Aybike , Lam Brian , Baranova Ancha , Younossi Zobair
Published: June 2021

A major complication of Hepatitis C Virus (HCV) is Insulin Resistance (IR), which consequently results in Diabetes Mellitus (DM). The aim of our study was to determine effects of HCV treatment on IR by monitoring three IR indicators in the serum: glucose, insulin, and c‐peptide. Patients diagnosed with genotype 1 HCV, were grouped by sustained viral response (SVR) into non‐responders and responders. Serum samples collected at the time of diagnosis and eight weeks into treatment were analyzed by ELISA for c‐peptide and insulin and by o‐toluidine reaction for glucose. Significant decrease was observed in the glucose concentrations in patients with SVR, whereas the non‐responder group showed a significant increase in insulin concentration. In addition, none of the non‐responders displayed a decrease in either C‐peptide or insulin levels over 8 weeks of treatment, whereas over half of the responder group showed improvement in both. These data offer promising evidence of a possible connection between SVR and IR. This study was partly supported by the Liver Disease Outcomes Fund of the Center for Liver Diseases at Inova Fairfax Hospital, and a Seed Grant from the Inova Health System.
The Role of Genetic Polymorphism of Beta‐3‐Adrenergic Receptor in The Susceptibility to Diabetes and Its Related Disorders
Authors: El Din Hemimi Neveen Salah , El Salam Mona Mohamed Abd , Tawfik Lamis M , Khalil Marwa Mahmoud
Published: June 2021

Background

The β3 – adrenergic receptor (β3 AR) is mainly expressed in adipose tissue and plays an important role in lipid metabolism and metabolic rate by mediating lipolysis and thermogenesis. It has been suggested that the Trp 64Arg (T→C) polymorphism in the β3 AR gene affects fat accumulation and/or impairment of lipid and carbohydrate metabolism.

Objective

To investigate if common polymorphism (Trp 64 Arg) of β3 AR gene, has a role in the apparent susceptibility to type 2 DM and its related disorders.

Material& Method

135 normal individual and 123 type 2 diabetic patients were enrolled in the study. The β3 AR Trp 64 Arg polymorphism was identified by restriction fragment length polymorphism polymerase chain reaction (RFLP‐PCR) of peripheral blood DNA samples. Analysis of data was done by using SPSS program.

Results

Allele frequency for C was 23.2% in the diabetic group compared to 12.2% in the control group. The carriers of XC genotype (TC and CC) were at high risk of developing type 2 DM (OR= 2.8; 95% CI = 1.6–4.9) when compared with the carrier of TT genotype. Furthermore they were at much higher risk of developing its related disorders such as central and abdominal obesity, dyslipidemia and hypertension (OR= 2.8; 1.8, 1.5, 2.2 and 2.7) for BMI, WHR, TG, HDL and hypertension respectively.

Conclusion

Arg 64 allele is a risk factor for DM and its related disorders.

Source of Fund: Ain Shams University.
Impact of type 1 diabetes (T1D) on vitamin B6 metabolism
Authors: Masse Priscille G. , Tranchant Carole C , Boudreau Jaclyn A , Ericson Karen , Ouellette Rodney
Published: June 2021

B6‐metabolites were analyzed in plasma (P), erythrocytes (E) and urine (U) of 22 insulin‐treated T1D patients without renal complication and 27 healthy controls in early adulthood (30–40 yrs). Groups were comparable due to rigorous selection criteria, excluding women on oral contraceptives, obese, vegetarian, pregnant or lactating. T1D (averaging 21 yrs) was relatively well‐controlled according to HbA1c. Kidney functions, assessed by U‐albumin/creatinine, did not differ between groups as well as U‐4‐pyridoxic acid (end‐metabolite), hemoglobin and serum albumin which were both adequate. P‐pyridoxal phosphate (PLP, indicator of B6‐nutritional status) was slightly below the cut‐off reference in T1D group. Its mean value and total B6‐aldehydes (PLP+PL) were not decreased significantly. Greater (p<0.01) alkaline phosphatase (ALP) activity in T1D group increased PL slightly (NS), resulting in inferior (p<0.05) P‐PLP/P‐PL ratio. ALP correlated strongly (p<0.001) with HbA1c. Elevated (NS) E‐PLP did not originate from pyridoxamine phosphate (unchanged), indicating no perturbation in B6‐dependent aminotransferase activity. Inferior (p<0.01) P‐PLP/E‐PLP ratio revealed a change between the two blood compartments. E‐PLP was highly (p<0.001) correlated with P‐PL. T1D impairs vitamin B6 metabolism through the elevation of ALP, dephosphorylating PLP to PL, the transfer form through cell membrane (P to E).
Long‐Extension PCR Studies of Mitochondrial DNA Deletions in Irradiated MOLT‐4 (Human Acute Lymphoblastic Leukemia) Cells and Pearson's Syndrome Fibroblasts
Authors: Lynch Zachary , Pogozelski Wendy
Published: June 2021

Mitochondrial DNA, a double‐stranded circle, undergoes large‐scale deletion events. In humans, many of these mitochondrial DNAs that bear deletions can accumulate and compromise energy production. Cells with high energy demands are particularly affected, leading to a variety of disorders. As a first step in an effort to determine how these large‐scale deletions arise, we have developed a long‐extension PCR assay that detects these deletions. Here we show the assay used to observe deletions in the mtDNA of γ‐irradiated human MOLT‐4 cells (a lymphoblastic cell line) as well as in a fibroblast cell line derived from a boy known to have elevated levels of a 4977‐bp deletion called the “common deletion”.

This research was sponsored by the Camille and Henry Dreyfus Foundation, the Geneseo Foundation and the SUNY Geneseo Student Association.
Effect of administration of ethanol extract of Eryngium carlinae on diabetic rats
Authors: Noriega‐Cisneros Ruth , Ortiz‐Avila Omar , Salgado‐Garciglia Rafael , Arellano‐Plaza Melchor , Clemente‐Guerrero Mónica , Manzo‐Avalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Diabetes mellitus is a metabolic disorder of multiple etiologies, characterized by chronic hyperglycemia, that is directly related to the development of long‐term complications which determine the quality of life of diabetic patients. At present is counted a variety of palliative medicine to treat this disease, however, some are beyond the reach of all people by their high cost.

Objective

The aim of this study was to investigate the effect of administration of the ethanol extract of Eryngium carlinae (EC) in diabetic rats.

Methods

Were measured glucose levels in blood and weight of the rats, likewise, was measured the food and water intake for 30 days, during which conducted oral administration (in drinking water) of the extract in a dose of 150 mg/kg. Diabetes was induced by a single injection (ip) of streptozotocin (40 mg/kg), rats with glucose levels above 300 mg/dL were used to shape the diabetic groups.

Results

The administration of extract of EC had no hypoglycemic effect; however, its use was able to reduce the weight loss characteristic of diabetic model. The symptoms of polyphagia and polydipsia were present in the diabetic group, as evidenced by the high consumption of food and water, compared with a no diabetic group (p <0.01) despite receiving treatment with extract.

Conclusion

Due to the results, currently being tested by varying the route of administration and dose of EC extract to find effective dose is also possible to evaluate the antioxidant activity of extract in different organs.
Regulation of pyruvate dehydrogenase kinase 4 (PDK4) by thyroid hormone: role of peroxisome proliferator activated receptor gamma coactivator (PGC‐1α)
Authors: Attia Ramy Raafat , Connaughton Sara , Wang Fang , Elam Marshall B. , Cook George A. , Park Edwards A.
Published: June 2021

Pyruvate dehydrogenase kinase 4 (PDK4) regulates pyruvate oxidation through the phosphorylation and inhibition of the pyruvate dehydrogenase complex (PDC). PDC catalyzes the conversion of pyruvate to acetyl‐CoA and is an important control point in glucose and pyruvate metabolism. PDK4 gene expression is stimulated by thyroid hormone (T3), glucocorticoids and long chain fatty acids. Here, we have identified two binding sites for the thyroid hormone receptor (TRß) in the promoter of the PDK4 gene. In addition, we have investigated the role of transcriptional coactivators and found that the peroxisome proliferator activated receptor gamma coactivator (PGC‐1α) enhances the T3 induction of PDK4. Addition of T3 to rat hepatocytes increased the abundance of the PGC‐1α and its association with the PDK4 promoter. Administration of T3 to hypothyroid rats increased PGC‐1α protein levels in the liver. In addition, we observed enhanced association of PGC‐1α not only with the PDK4 gene but also with phosphoenolpyruvate carboxykinase (PEPCK) and carnitine palmitoyltransferase 1a (CPT‐1a) genes. Knock‐down of PGC‐1α in rat hepatocytes reduced the T3 induction of PDK4, PEPCK and CPT‐1a genes. Our results indicate that T3 regulates PGC‐1α abundance and association with hepatic genes and in turn PGC‐1α is an important participant in the T3 induction of selected genes.
Histone H4 lysine‐16 acetylation regulates cellular lifespan
Authors: Dang Weiwei , Steffen Kristan K , Kaeberlein Matt , Kennedy Brian K , Berger Shelley
Published: June 2021

Cells undergoing developmental processes are characterized by persistent non‐genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post‐translational modifications. Sirtuins, a group of conserved NAD+‐dependent deacetylases or ADP‐ribosylases, promote longevity in diverse organisms; however, their molecular mechanisms in aging regulation remain poorly understood. Yeast Sir2, the founding member of the family, establishes and maintains chromatin silencing by removing H4 lysine 16 acetylation and bringing in other silencing proteins. Here we show an age‐associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing yeast aging models and the role of Sir2 in controlling the accumulation of extra‐chromosomal rDNA circles (ERCs) during aging, may represent an evolutionarily conserved function of Sirtuins in regulation of replicative aging by maintenance of intact telomeric chromatin.
Relative tissue expression of homologous torsins can explain the neuronal specific importance of DYT1 dystonia‐associated torsinA
Authors: Jungwirth Michael Thomas , Dear Mary Lynn , Brown Patricia , Holbrook Kristen , Goodchild Rose Ellen
Published: June 2021

A deletion in the widely expressed TOR1A gene causes the childhood onset, neurological disease of DYT1 dystonia. Mouse Tor1a gene knock‐out also specifically affects the developing nervous system. However, in both cases, the basis of tissue specificity is unknown. TOR1A is one of four predicted mammalian torsin AAA+ proteins (ATPases associated with Assorted cellular Activities), raising the possibility that a homologous torsin operates in non‐neuronal tissues. We find that all four torsins reside in the endoplasmic reticulum lumen. In addition, multiple torsins are present in high Mr complexes, consistent with assembly into an oligomeric AAA+ enzyme. Introducing a mutation (WBEQ) that typically stabilizes AAA+ proteins in a substrate‐bound state causes several torsins to associate with a shared nuclear envelope binding partner. In addition, this association requires the torsinA interacting protein, LAP1. Most torsin proteins are widely expressed. However, we identified that embryonic neuronal tissues contain relatively low amounts of torsinB, therefore revealing that torsinB expression inversely correlates with the cell and developmental requirement for torsinA. In conclusion, our data indicate that many cell types express torsin AAA+ proteins and that differential torsinB expression may contribute to both the neuronal specific importance of torsinA and the symptom specificity of DYT1 dystonia.
AMPK Targets: Predicted by Mass Spectroscopy and Proteomic Sequence Mapping
Authors: La Nai Marin Traci , Gongol Brendan Richard , Shyy John Y‐J.
Published: June 2021

AMP activated protein kinase (AMPK) plays an important role in regulating energy homeostasis via its serine/threonine kinase activity. This project aimed to identify putative novel AMPK targets in effort to expand currently known biological signaling pathways involving AMPK. Cell lysates collected from wild‐type murine embryonic fibroblasts (MEFs) treated with AMPK activator, 5‐aminoimidazole‐4‐carboxamide1‐β‐d‐ribonucleoside (AICAR) or MEFs ablated for AMPKα1 and α2 were analyzed by Liquid Chromotography/Mass (LC/MS). Three data sets were used to identify potential targets of AMPK and their putative phosphorylation sites (MASCOT score ≥35, p value <0.005). The initial bioinformatic screen utilized an in‐house generated R program script to map an accepted AMPK phosphorylation consensus recognition motif from ENSEMBL proteomes. Scansite was recruited for a second bioinformatic screen to map a less stringent consensus sequence to SWISS‐PROT proteomes. From these 5 data sets, 812 proteins contain the most stringent consensus motif, 3,158 contain a less stringent consensus sequences, 686 were present in 2 or more of the screens, and 14 were present in at least 4 screens. Of the obtained target sequences, 24 are known to be phosphorylated by AMPK. Thus, LC/MS in conjunction with bioinformatics may provide an efficient high throughput screening for predicting kinase substrates.
Pin1 activates gonadotropin β‐subunit gene transcription through its interaction with specific transcriptional factors
Authors: Luo Zhuojuan , Wijeweera Andrea , Oh Yingzi , Liou Yih‐Cherng , Melamed Philippa
Published: June 2021

Pin1 is a peptidyl‐prolyl cis/trans isomerase which catalyzes the isomerisation of phosphorylated Ser/Thr‐Pro peptide bonds. Pin1 knock‐out mice have a phenotype showing marked abnormalities in their reproductive development and function. However, the molecular mechanisms underlying their reproductive defects are poorly understood. We demonstrate here that Pin1 is required for the basal and GnRH‐induced gonadotropin β‐subunit gene transcription. Knocking down Pin1 expression by small interfering RNA (siRNA) reduces both gonadotropin β‐subunit gene promoter activity and endogenous mRNA levels. In addition, co‐immunoprecipitations and mammalian two hybrid assays show that Pin1 interacts with the transcription factors, SF‐1, Pitx1 and Egr‐1. Pin1 activates transcription of the gonadotropin β‐subunit genes synergistically with these transcription factors, either by modulating their stability or through increasing their protein‐protein interactions. Our results suggest that Pin1 functions as a novel player in GnRH‐induced signal pathway, involved in gonadotropin β‐subunit gene transcription by modulating the activity of various specific transcription factors.
The Role of TBX3 in the Regulation of Tumor Suppressor p14ARF Expression in Human Liver Carcinoma Cells
Authors: Liang Lidia tian , Huang Taosheng
Published: June 2021

TBX3 is a transcription factor in the T‐box gene family. Mutations of TBX3 in humans are associated with Ulnar‐Mammary syndrome, an autosomal dominant condition with multiple organ abnormalities. The recent studies showed that overexpression of TBX3 is associated with breast cancer, probably by interacting with HDACs and inhibiting p14ARF. The tumor suppressor p14ARF is a vital biomarker for cancer development. To determine if the misregulation of TBX3 gene expression could disrupt the expression of tumor suppressor p14ARF in human liver carcinoma cells, we established an inducible expression of the TBX3 cell model with the Tet‐off Advanced System. The results showed that the expression of endogenous p14ARF in human liver carcinoma cells was suppressed by TBX3 in a dose‐dependent manner. This experiment will facilitate our understanding of the molecular mechanisms underlying the TBX3‐initiated liver tumorigenesis, as we as provide potential for liver cancer diagnostics and therapeutics.
POLY(ADP‐RIBOSE) POLYMERASE‐1 IS A DETERMINING FACTOR IN CRM1‐MEDIATED NUCLEAR EXPORT AND RETENTION OF P65 NF‐κB UPON TLR4 STIMULATION: A NOVEL MECHANISM OF REGULATING NF‐κB‐DEPENDENT GENE EXPRESSION
Authors: Mourad Zerfaoui , Errami Youssef , Naura Amarjit S , Suzuki Yasuhiro , Boulares Hamid
Published: June 2021

The mechanism by which PARP‐1 affects NF‐κB activation has been elusive. Here, we show that PARP‐1 knockout prevented translocation of p65 NF‐κB to nuclei of smooth muscle cells (SMCs) upon TLR4 stimulation by LPS and subsequent iNOS and ICAM‐1 expression. Such defects were reversed by reconstitution of PARP‐1 expression. PARP‐1 expression was dispensable for I‐kBα phosphorylation and subsequent degradation but was required for p65 NF‐κB phosphorylation in LPS‐treated SMCs. p65 NF‐κB exhibited perinuclear localization in a subpopulation of LPS‐treated PARP‐1 knockout cells suggesting a nuclear import defect. However, expression of importin α3 and importin α4, the two proteins necessary for NF‐κB nuclear import, and their cytosolic localization were unaltered in LPS‐treated PARP‐1 knockout cells. Inhibition of Crm1 by leptomycin‐B promoted p65 NF‐κB nuclear accumulation and phosphorylation in PARP‐1 knockout cells as well as reversal of iNOS and ICAM‐1 expression only upon TLR4 stimulation. p65NF‐κB poly(ADP‐ribosyl)ation decreased its interaction with Crm1 in vitro suggesting that such an effect may be critical for the interaction and may constitute a determining factor in nuclear retention of p65 NF‐κB upon TLR4 stimulation. These results provide novel insights into the mechanism by which PARP‐1 promotes NF‐κB nuclear retention and dependent gene expression.
Structural and functional characterization of the core single‐stranded DNA‐binding domain of purine‐rich element binding protein B (Purβ)
Authors: Rumora Amy E. , Ramsey Jon E. , Ballif Bryan A. , Kelm Robert J.
Published: June 2021

Purβ is a sequence‐specific single‐stranded DNA (ssDNA)‐binding protein that represses smooth muscle α‐actin gene transcription in stress‐activated fibroblasts and smooth muscle cells via cooperative interaction with two sites in the purine‐rich strand of a 5′ enhancer element (dubbed PE32‐F). To better understand how the modular structure of Purβ dictates its specific ssDNA‐binding function, we evaluated the interaction of purified recombinant Purβ with PE32‐F using spectroscopic and biochemical approaches. Results of intrinsic protein fluorescence and circular dichroism analyses pointed to a ssDNA‐induced change in protein conformation. Consistent with this interpretation, Purβ was protected from proteolysis when complexed with PE32‐F. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment spanning residues 29‐305 as determined by mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to dimerize. Quantitative ssDNA‐binding assays revealed that the core domain binds to PE32‐F with similar specificity but increased affinity compared to full‐length Purβ and a truncated 37‐263 mutant absent a putative amphipathic helix‐forming module. These findings provide new insight into the structural basis for the specific and high affinity binding of Purβ to ssDNA. NIH R01 HL054281, T32 HL007594, P20 RR16462.