bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of cyclin E stability in Xenopus embryos
Authors: Brandt Yekaterina , Mitchell Therese , Wu Yuehan , Hartley Rebecca 
Published: June 2021

Cyclin E associates with Cdk2 to mediate the G1/S cell cycle transition and is overexpressed in many cancers. We are studying how cyclin E is downregulated in Xenopus laevis embryos to ask if similar mechanisms are disrupted during tumorigenesis. In Xenopus embryos, cyclin E level is constant until its abrupt destabilization by an undefined mechanism after the12th cell cycle, corresponding to midblastula transition (MBT). Degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent. To examine the role of phosphorylation in cyclin E turnover in Xenopus, we generated a series of threonine/serine mutations. Western blot analysis showed that mutation of threonine 394 to alanine (T394A) and both T394A/S398A increased cyclin E stability. T75A mutation had only a minor effect. Immunofluorescence analysis showed that cyclin E re‐localized from the cytoplasm to the nucleus preceding degradation. When cyclin E nuclear import was inhibited, its stability was markedly increased after MBT. We conclude from these studies that cyclin E destruction is facilitated by phosphorylation and requires nuclear import. Treatment of embryos with a proteasome inhibitor caused progressive cytoplasmic accumulation of cyclin E after MBT, suggesting that it may be exported back to the cytoplasm for destruction by the proteasome. This work was supported by NIH grant R01CA095898 to RSH.Grant Funding Source: NIH
Stimulation of adrenergic receptors regulates proteasome function in cardiomyocytes
Authors: Ranek Mark John , Wang Xuejun 
Published: June 2021

Ubiquitin‐proteasome system (UPS)‐mediated proteolysis plays a central role in protein quality control and degradation in the cell. UPS dysfunction was observed in various forms of heart disease and might play an important role in the development of congestive heart failure (CHF). However, very little is known about how UPS proteolytic function is regulated in the heart.The sympathetic nervous system (SNS) activity is increased during CHF. As an initial step to investigate if and how the SNS regulates UPS function in the heart, we employed neonatal rat ventricular myocytes (NRVM) culture. To monitor UPS function, a validated UPS surrogate substrate (a modified Green Fluorescent Protein, GFPu) and a red fluorescent protein (RFP, not a specific substrate of the UPS) were co‐expressed in cultured NRVMs through adenoviral gene deliveryWe found that both stimulation of the beta 1 receptor with dobutamine and stimulation of the alpha 1 receptor with phenylephrine significantly reduced the GFPu/RFP ratio, suggesting an enhancement of UPS function. We have also discovered that the phosphorylation status of the Rpt6 subunit of the 19S proteasome and the beta 5 subunit of the 20S proteasome was markedly altered by stimulating each of the receptors. These preliminary findings suggest that SNS may play an important role in regulating cardiac proteasomes. Funding: NIH HL085629, HL072166, and HL068936.
A comparative study of the lipid composition of the brain of chicken and rat during myelination; A chromatographic‐densitometric analysis.
Authors: Helmy Fatma Mahmoud , Morris Aaron 
Published: June 2021

The lipid profile of brain from 10 day and 18 day old chick embryo, one day old chick and adult chicken (10 weeks old), as well as full‐term fetus rat, 21 day old male rat, young adult male rat, and pregnant female rat brain; were analyzed by thin layer chromatography and densitometry.10‐day old chick embryo brain revealed a low concentration of GDG, n‐CMH, h‐CMH, and CMH‐S. The concentration of these glycolipids increased gradually as the chick embryo advanced in development, indicating age relatedness, and reached the highest level in the brain of adult chicken. Rat brain did not begin to show the presence of these lipids until at least 21 days old, possibly indicating that the nervous system of the bird model develops faster than that of mammals. In addition, phosphatidyl ethanolamine plasmalogen (PE), was shown to be the only alkenyl phospholipid in all samples analyzed. Two molecular species have been identified (PE1 and PE2), and PE2 has been shown to correlate with the myelination process. Phosphatidyl choline (PC), and sphingomyelin (SM), were also present. SM concentration increased gradually during development, and reached its highest level in adult chicken brain, as in rat brain.The correlation of myelination, brain development and the level of these glycolipids, indicated the important role of these glycolipids in both brain maturation and function.
Characterization of Multicopper Oxidase Related Protein (MCORP) in Two Insect Species
Authors: PENG ZEYU , Gorman Maureen J , Arakane Yasuyuki , Kanost Michael R 
Published: June 2021

Multicopper oxidases (MCOs) have diverse functions but are evolutionarily and structurally related. Ten histidines and one cysteine form two highly conserved copper binding centers, which are required for oxidase activity. We discovered a gene that we named multicopper oxidase related protein (MCORP), which looks somewhat like a MCO, but lacks the conserved cysteine and many of the histidines. We identified MCORP orthologs in many insect species, including Tribolium castaneum, Anopheles gambiae, Aedes aegypti, Culex pipiens, Bombyx mori, and Acyrthosiphon pisum. Phylogenetic analysis of MCORPs and MCOs indicated that MCORPs belong to a specific cluster. We cloned full‐length cDNAs of A. gambiae and T. castaneum MCORP and studied their expression patterns by RT‐PCR. Both genes are highly expressed in late developmental stages, particularly in pupae. In adults, AgMCORP is expressed predominantly in reproductive tissues. We purified recombinant AgMCORP and TcMCORP to make antibodies that we will use to determine the location of MCORP within an insect. RNAi mediated knock down of MCORP in T. castaneum resulted in a pupal lethal phenotype, which demonstrated that TcMCORP is essential to pupal development. To try to learn the function of MCORP, we will expand our RNAi studies and determine whether recombinant MCORPs have enzymatic activity.This research was supported by a grant from NIH (R01 AI070864).
Effect of TBT exposures on Protein kinase D activity of human Natural Killer cells
Authors: Rana Krupa , Whalen Margaret M. 
Published: June 2021

Natural killer (NK) cells are lymphocytes that target and destroy (lyse) tumor cells, virally infected cells, and antibody coated cells. Due to their capacity to respond without a need for prior sensitization, they are at the forefront of the immune response to tumor and virally infected cells. Tributyltin (TBT) is an organotin that is found in many different products. It is found in antifouling paints and has been used as an antifungal agent in a wide variety of applications including silicon baking parchments and shower curtains. Due to its contamination of the environment it has also been found in human blood. Exposure of human NK cells to TBT decreases their lytic function and causes activation of mitogen activated protein kinase (MAPK). The aim of the current study is to examine the role of the serine threonine kinase, protein kinase D (PKD) in the TBT‐induced activation of NK cell MAPKs. PKD activation has been shown in other cells types to lead to the activation of MAPKs. In this study NK cells were exposed to 300, 200, 100, 50, and 25 nM TBT for 10 min and 1 h and cell lysates were prepared and analyzed by SDS‐PAGE followed by Western blot. The activation state of PKD was monitored using an antibody to phospho‐PKD. The results indicated that PKD was activated approximately two fold by 300‐100 nM TBT within 10 min. These results suggest that TBT‐induced activation of PKD may be in part responsible for the TBT‐induced activation of MAPK.Supported by NIH grant S06 GM008092‐34.
Decreased mineralization and lack of cyclin D1 response to Parathyroid Hormone Related Peptide (PTHrP) in primary osteoblasts isolated from MKP‐1 knock out mice
Authors: Datta Nabanita , Chaker Bayan , Abou‐Samra Abdul 
Published: June 2021

MAPK phosphatase 1 (MKP‐1), a member of the dual‐specificity phosphatases, inactivate MAPKs via dephosphorylation of ERK, p38 and JNK. Whether MKP‐1 plays a role in PTH and PTHrP anabolic action in bone is yet to be investigated. Our previous study demonstrated a decrease in ERK‐MAPK and cyclin D1 and an increase in MKP‐1 expression in differentiated osteoblasts following PTH/PTHrP induction both in vitro and in vivo. To investigate the effect of MKP‐1 deletion in PTH1R anabolic action, primary calvarial osteoblasts were isolated from neonatal or 10‐week old MKP‐1 knock out (KO) and wild type (WT) mice via sequential collagenase digestion. The cells were differentiated with ascorbic acid (100μg/ml) and β‐glycerophosphate (5mM) and harvested between 7 to 21 days. Von Kossa staining at day 14 showed decreased mineralization in MKP‐1 KO osteoblasts compared to WT. In contrast to WT cells incubation of MKP‐1 KO osteoblasts with 100nM PTHrP had no effects on pERK or cyclin D1 expression. MicroCT analysis of mid‐diaphyses of tibiae and femora from 2 week old MKP‐1 KO mice showed decreased bone mineral density (BMD), bone mineral content (BMC) and tissue mineral content (TMC) compared to WT bone. Taken together, these results show that MKP‐1 may be important for PTH/PTHrP anabolic action in osteoblasts and play a role in the maintenance of bone mass.(Supported by NIH R03 DE018245‐01 and R01DK53904)
S‐Adenosylmethionine (SAM) downregulates phosphodieterease 4B expression and attenuates endotoxin‐induced TNF expression in monocytes via cAMP/PKA pathway
Authors: Gobejishvili Leila , Barve Shirish , Kirpich Irina , Avila Diana , Joshi‐Barve Swati , McClain Craig 
Published: June 2021

Elevated concentration of circulating TNFα occurring due to leaky gut and endotoxemia plays a significant role in the pathogenesis of several acute and chronic inflammatory disorders. Although SAM attenuates endotoxin stimulatable TNF production in vitro and in vivo, the underlying mechanisms are not fully understood. Our earlier work demonstrated that SAM decreases LPS‐inducible TNFα gene expression and attenuates organ injury. Further, our recent studies showed that PDE4B/cAMP pathway plays a critical role in the regulation of LPS‐inducible TNF expression. Accordingly, in the present study PDE4B expression was examined as the potential inflammatory mechanism targeted by SAM.The results obtained demonstrate for the first time that exogenous SAM supplementation significantly lowered LPS‐inducible PDE4‐specific activity, increased cellular cAMP levels and consequently decreased LPS‐inducible TNFα expression. Further, the SAM/PDE4/cAMP effect on TNF expression was partially PKA dependent. Notably, these data identify PDE4 expression as a significant pathogenic component of endotoxin mediated inflammation which can be targeted by SAM for the potential therapeutic use in the treatment of diseases characterized by elevated TNFα expression.This work was supported by NIH grants AA014371, AA015970, and Office of Dietary Supplements.
Analysis of ESTs for alternatively spliced 3′ and 5′ splice sites reveals optimal exon size in vertebrates
Authors: Shepard Peter Joseph , Hertel Klemens J 
Published: June 2021

In vertebrates most exons are small surrounded by much larger introns. Due to this architecture, splice sites are initially recognized across the exon in what is known as exon definition. Here we examine the relative usage of 3,000 pairs of alternative 3′ splice sites and 2,000 pairs of alternative 5′ splice sites in the human genome. We find that within an optimal exon length window >50 <250 nts, there is a positive correlation between splice site strength and splice site usage. However, when one splice site is located inside of this exon length and the other is not, the splice site inside of the optimal exon length is preferred regardless of splice site strength. Our analysis reveals that splice sites within an optimal exon size are preferentially used when compared to splice sites outside of this optimal size. We conclude that a constraint of exon size is a fundamental parameter that dictates splice site usage. Because the spliceosome eventually forms across the intron, it is believed that intron definition is the predominant mode of splice site recognition for introns smaller than 250 nucleotides in length. However, in a subset of our alternative splice sites, in which one splice site is exon defined and the other is intron defined, the exon defined splice site is preferentially used, suggesting that exon recognition is the default mode of splice site recognition in vertebrates.NIH
Vitamin A and vitamin E levels in lung from subjects with chronic obstructive pulmonary disease with different degree of severity of emphysema
Authors: Schäffer Michael W , Roy Somdutta Sinha , Das Salil K 
Published: June 2021

Vitamin A (VA) and its active metabolites play an essential role in the respiratory tract by influencing differentiation and the integrity of the epithelium. A study that attempted to relate VA status with the degree of lung airway obstruction, revealed a lower serum retinol concentration in patients with moderate to severe COPD. Further, treatment with retinol improved the FEV1 in subjects with COPD.To test our hypothesis that VA signaling pathway is compromised in COPD, we analyzed human lung samples from COPD patients, differing in the degree of severity of emphysema, provided by the Lung Tissue Research Consortium for their VA and tocopherol (T)‐levels by HPLC. T levels were determined since it is known that α–T is an inhibitor of retinyl ester hydrolase activity. VA levels (retinol and retinyl ester) were found to decrease with severity of the disease from mild (168 ng/g, 160 ng/g), to moderate (136 ng/g, 99 ng/g), and to severe (127 ng/g, 71 ng/g). α‐T levels were less in moderate (12.8 μg/g) and severe (13.7 μg/g) groups than that in mild group (15.5 μg/g). Similarly δ‐T‐levels were less in moderate (229 ng/g) and severe (142 ng/g) groups than that in mild group (231 ng/g). However, it is interesting to note that γ‐T levels were higher in moderate (4.6 μg/g) and severe (2.2 μg/g) groups than that in mild group (1.3 μg/g). Results indicate an involvement of VA & Ts in COPD. (Supported by NIH grant 1RO3 HL095419‐01A1).
Sox transcription factor mediated transcriptional regulation of Robo4 expression and function
Authors: Samant Ganesh Vinayak , Pramanik Kallal , Leigh Noah , Horswill Mark , Beltrame Monica , Ramchandran Ramani 
Published: June 2021

Our laboratory has identified Roundabout4 (Robo4) as a bonafide angiogenic receptor. Robo4 knockdown (KD) endothelial cells show decreased migration and zebrafish (zf) embryos show intersomitic vessel (ISV) defects. We have reported that Robo4 affected cell migration results in imbalanced active Cdc42 and disrupted actin organization but precise molecules that interact with Robo4 remain unknown. To identify the proteins that bind and orchestrate Robo4 mediated angiogenesis, we performed Yeast 2 Hybrid (Y2H) screen with cytoplasmic zf Robo4 as bait and zf cDNA as prey. Y2H identified many SOX transcription factor (TF) proteins as potential binding partners. Based on their vascular specificity we focused on Sox18 and Sox7 and immune precipitation confirmed that Robo4 binds to both. In vivo, Robo4 KD in conjunction with Sox18 and Sox7 KD decreased Fli ISV expression, indicating disrupted angiogenesis. Bioinformatics on Robo4 promoter reveals Sox TF binding sites and signifies transcriptional regulation of Robo4 via Sox. Using transactivation assays we have identified that Sox18/7 temporally induce Robo4 promoter. Sox18/7 double KD decreases Robo4 transcription and expression in zf vasculature. In sum, our data indicate that Robo4 and Sox TF interact genetically and Sox TF mediated transcriptional and post‐translational Robo4 expression and function is critical in angiogenesis.Grant Funding Source: NIH
Endothelial sprout formation during vasculogenesis
Authors: Czirok Andras , Szabo Andras , Kosa Edina , Little Charles D , Lansford Rusty , Rupp Paul A 
Published: June 2021

We studied vasculogenic sprouting, the multicellular patterning mechanism of vasculogenesis, with time‐lapse microscopy in avian embryos, and in cultures of endothelial cells invading collagen I gels. Cell‐autonomous motility and tissue movements were distinguished utilizing digitally filtered displacements of various immuno‐labeled extracellular matrix components. Endothelial cell motility was investigated inside the forming vessels through a recently developed TIE1‐YFP transgenic quail line. Displacements of pulse‐labeled fibronectin fibers around the nascent vessels reveal that the fibronectin accumulated is of mesodermal origin, and additional filaments are not assembled de novo during the process. Correlational analysis of endothelial cell movements and the morphology of fibronectin filaments shows that fibronectin does not act as a vascular pre‐pattern to guide multicellular sprouts. Sprout elongation depends on a continuous supply of endothelial cells, streaming along the sprout towards its tip. With computer simulations we explore the conditions when cell‐cell guided active cell motility can result in multicellular streaming and sprouting behavior.Grant Funding Source: NIH R01HL087136
Staphylococcus Enterotoxin B induced Host Response Signatures in the Presence and Absence of Battlefield‐like Stress
Authors: Muhie Seid , Hammamieh Rasha , Yang David , Jett Marti 
Published: June 2021

In previous studies, we identified transcripts and proteins suppressed by battlefield‐like stress that were essential for protective immunity. In this study, pre‐ and post‐training leucocytes, from US Army Ranger Trainees, were exposed in vitro to Staphylococcal enterotoxin B (SEB) to analyze SEB induced regulation of gene expression in the presence and absence of severe battlefield‐like stress. cDNA microarrays and real time PCR arrays were used for transcriptome wide profiling, and computational analyses to identify regulatory modules. Stress‐suppressed immune response genes were remained suppressed in SEB treated post‐training leukocytes. Despite complications due to suppressed immune responses seen at the end of intensive battlefield‐like training, we have identified other transcripts that showed consistent differential expression towards SEB in both pre‐ and post‐training leukocytes even in comparison to dengue virus and Yersinia pestis exposure. Ontological and regulatory network analyses revealed that SEB‐specific transcripts are involved in heparin binding, positive regulation of mitochondrial depolarization, apoptosis, negative regulations of JAK‐STAT cascades and axonogenesis. Identified SEB signatures have the potential to be used as SEB‐exposure markers under battlefield stress.We are grateful to DTRA for funding.
Causal directions in the relationship between perinatal depressive symptoms and breastfeeding difficulties
Authors: Vitta Bineti Sidi , Nommsen‐Rivers Laurie A , Cohen Roberta J , Chantry Caroline J , Dewey Kathryn G 
Published: June 2021

Perinatal depressive symptoms (DS) are associated with shorter breastfeeding duration, but the causal direction is not clear. Among 415 Californian primiparae who initiated breastfeeding, we examined if 1) prenatal DS are associated with greater risk for breastfeeding difficulties, and 2) breastfeeding difficulties are associated with worsening DS between pregnancy and day 14 (d14) postpartum (pp). Adjusting for socio‐demographic variables and prenatal breastfeeding intentions (logistic regression adjusted odds ratio [95% confidence interval]), women with prenatal DS were more likely to experience nipple pain in the first 24h pp (2.06[1.16–3.65]), between 1–2wk pp (2.27[1.14–4.52]) and 2–4wk pp (2.13[1.03–4.42]); to have babies who were suckling poorly on d7 pp (1.93[1.06–3.51]); and to supplement with formula between 1–2mo pp (2.06[1.14–3.73]). Worsening of DS between pregnancy and d14 pp was associated with poor infant suckling on d7 pp (2.08[1.26–3.42]) and formula use between d3–7 pp (1.63[1.06–2.50]). Thus, the relationship between perinatal depressive symptoms and breastfeeding difficulties may be bidirectional with prenatal DS leading to nipple pain, poor infant suckling, and formula supplementation; and poor suckling and early formula supplementation leading to worsening DS.Supported by a grant from the Department of Health and Human Services, Maternal Child Health BranchGrant Funding Source: Department of Health and Human Services, Maternal Child Health Branch
Genistein mediates perturbations in one‐carbon metabolism during diet‐induced folate deficiency
Authors: Nonnecke Eric B , Schalinske Kevin L 
Published: June 2021

Populations consuming isoflavone‐rich diets exhibit lower prevalence of heart disease and estrogen‐responsive cancers. Genistein, a17β‐estradiol‐like isoflavone, has been shown to modify the fetal epigenome in utero in the agouti mouse model and modulate DNA methylation patterns in estrogen‐responsive tissues. Moreover, it also exhibits antiproliferative action in cancerous cells via suppression of S‐adenosylmethionine –dependent DNA methyltransferases, leading to site‐specific hypomethylation. However, it is not known if genistein modulates one‐carbon metabolism, a process that when disrupted leads to dysregulation of the epigenome. The objective of this study was to characterize the metabolic actions of genistein during moderate dietary folate deficiency. Male Sprague Dawley ratswere fed an ad lib control or folate‐deficient (FD) diet with or without genistein supplementation (300 mg/kg diet) for 8 wk. Plasma homocysteine (Hcy) concentrations in genistein‐supplemented, FD animals (16.7 μM) was lower (P = 0.02) than the Hcy concentration of FD animals not supplemented with genistein (27.9 μM). Hepatic enzyme analysis suggests the ability of genistein to attenuate hyperhomocysteinemia in FD rats is not linked to diminished Hcy production. Clearly, genistein has the ability to modulate Hcy and methyl‐group metabolism; however, the underlying mechanisms remain unknown.Grant Funding Source: AHA assn
Effect of Physiological Levels of Sodium Selenite on the Expression and Regulation of Hypermethylated Tumor Supressor Gene in Human Breast Cancer Cell Line
Authors: Chen Yu‐Hsin , Yang Feili Lo , Kao Cheng‐Fu , Chen Yaling 
Published: June 2021

Breast cancer (BC) was the 4th‐leading cause of cancer death in Taiwan with the rising incidence as malignant type in young premenopausal women during the recent decades. The aim of our study was to investigate the effects of physiological levels of selenite on regulation of BC tumor suppression gene through hypermethylation. Exposure of human breast cancer cell line MDA‐MB 231 to 1.5 and 2.5 μM of sodium selenite for 7 days significantly decreased cell invasion and migration ability compared to control. Selenite treatment (2.5 μM) reactivated the anti‐invasion gene CST6 (cystatin E/M), of which the promoter region hypomethylation was confirmed by methylation‐specific PCR. The protein expression of DNA methyltransferase £L (DMNT1) and histone deacetyl transferase I were also significantly decreased by selenite treatment. In conclusion, the suppressed invasion ability of MDA‐MB 231 cells by physiological levels of selenite may be due to the suppressed protein expression of DNMT1 as the result of reactivating the anti‐invasion genes such as CST6, of which the promoter region were hypomethylated. The results of this study are expected to explain the role of selenium in regulating the re‐expression of specific tumor suppressor genes and to be applied in improving the prognosis of breast cancer.(NSC98‐2320‐B‐030‐004)Grant Funding Source: NSC98‐2320‐B‐030‐004