bluuebunny/crossref_metadata_2025 huggingface dataset:
The alpha‐galactosyl derivatives of the ganglioside GD1b are essential for maintenance of structure and organization in mast cells
Authors: Oliver Constance , Silveira e Souza Adriana Maria Mariano , Orlandini Rodrigo de Castro , Trindade Edvaldo da Silva , Jamur Maria Célia
Published: June 2021

The exact function that gangliosides play in cell physiology is poorly understood. In this study, the role of mast cell specific gangliosides derived from GD1b in maintaining cell structure was examined using RBL‐2H3 cells and two mutant mast cell lines (E5 and D1) derived from them. The two deficient cell lines were morphologically distinct from each other as well as from the parental cells. Actin filaments in RBL‐2H3 and E5 cells lay under the plasma membrane following the spindle shape of the cells, whereas in D1 cells the filaments were concentrated in large membrane ruffles. Microtubules in RBL‐2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum was vesiculated in the mutant cell lines. The Golgi complex was disorganized in the E5 and D1 cell lines and the secretory granule matrix in the mutant cells was altered. These results suggest that the mast cell‐specific α‐galactosyl derivatives of ganglioside GD1b are important in maintaining normal cell morphology. Financial Support: FAPESP, CAPES, CNPq, FAEPA.
Evidence of microscopic clusters of neuronal cell bodies along the spinal accessory nerve
Authors: Hovorka Michelle S. , Uray Nandor J.
Published: June 2021

Our objective was to examine the accessory nerve for the presence of microscopic clusters of sensory neuronal cell bodies. This study was prompted by recent articles which show a few cases of a grossly visible ganglion along a communication between the spinal accessory nerve and the C1 spinal nerve. Our research has not found any studies that have indicated previous findings of microscopic clusters of neuronal cell bodies. We dissected the upper cervical spinal cord, nerve roots, and accessory nerve out of embalmed cadavers. The accessory nerve was separated from the rest of the tissue and serially sectioned. Histological examination of the cervical contribution of the accessory nerve consistently shows clusters of neuronal cell bodies along it, thus constituting a ganglion, although not grossly discernable. Funding is provided by the Kirksville College of Osteopathic Medicine Graduate Program.

Grant Funding Source

Kirksville College of Osteopathic Medicine Graduate Program
Viability of estrogen and lithium‐treated primary brain cell cultures following glutamate insult and NR1 mRNA expression of pretreated cultures
Authors: Valdes James Jason , Weeks Ophelia Inez
Published: June 2021

Glutamate is an excitatory neurotransmitter that facilitates calcium influx, and excess calcium influx is detrimental to brain cells. Excitotoxicity is implicated in many neurological diseases and estrogen (E2) and lithium can influence excitotoxicity inversely: E2 facilitates calcium influx, while lithium impedes this influx. We harvested cortical and hippocampal cells of C57BL/6J fetal mice and pre‐treated for 48 hours with E2, lithium or combined E2/lithium. Cultures were then exposed to a toxic level of glutamate (100μM). Cell viability using fluorescein diacetate/propidium iodide assay revealed that only lithium pretreatment increased viability of cortical cultures and combined pretreatment did not. All pretreated hippocampal cultures reduced cell viability. Total RNA was also isolated from pre‐treated cultures and reverse transcribed. cDNA was then amplified via PCR using a glutamate receptor subunit (NR1), neuron and glia gene‐specific primers. Combined E2/lithium treatment reduced NR1 mRNA expression for both culture types. Additionally, these cultures expressed increased glial fibrillary acidic protein (GFAP) mRNA while neurofilament‐heavy chain (NF‐H) mRNA was low. Our data suggest lithium may protect against glutamate insult, but combined doses of E2 and lithium interfere with/prevent protection against glutamate excitotoxicity and NR1 mRNA expression among these cultures.
The blood supply of the peripheral branch of the facial nerve
Authors: Tamatsu Yuichi , Ishibe Koji , Shimada Kazuyuki
Published: June 2021

Because of the long extension of axon aiming to distal region, it is necessary for all peripheral nerves to derive the nutrient supply from not only the nerve cell body but also the peripheral part. When the peripheral nerve passes, In general, nutrient artery "Vasa nervorum" accompanies nerve fiber to peripheral region and derives in the epineurium, reach the endoneurium through the perineurium and make capillary plexus. It is also said Vasa nervorum of facial nerve concerns facial nerve palsy. Therefore the morphological features of the blood supply of the facial nerve out of the stylomastoid foramen were observed. Eighteen adult cadavers from 46 to 80 years old in whom dye was injected were observed under stereo microscope. Moreover, the region of the epineuria was observed with SEM. As a result, Vasa nervorum was derived from complex reticulation structure that were formed of mainly superficial temporal, facial, transverse facial and zygomatico‐orbital artery and collaterally supraorbital, deep temporal, buccal artery and parotid branches. SEM showed that one capillary was accompanied in each perineurium.
MRI Assessment of Amygdalar Size Based on a Single Plane Measurement in Patients with Clinical Diagnosis of Alzheimer's Disease and Mild Cognitive Impairment
Authors: Brabec Jiri , Horinek Daniel , Krasensky Jan , Seidl Zdenek , Rulseh Aaron , Hort Jakub , Petrovicky Pavel
Published: June 2021

MRI volumetry of the amygdala is a relevant marker of the severity of dementia in Alzheimer's disease. To evaluate the changes of amygdalar volume, there are many manual tracing techniques, which are time‐consuming for clinicians. On the basis of our previous studies, we developed an easy method which allows estimation of the total volume of the amygdala from a simple referential section (frontal section in the level of the anterior pole of the hippocampus). Boundaries of the amygdala in this level are clear and can be easily distinguished from neighboring structures. This protocol was developed with a sample of 70 healthy brains (anatomical preparations and MRI's of healthy volunteers). The average area of the sections through the amygdala in the reference level was 1.64 cm2 (SD=0.23). In normal brains, the referential section correlated highly with the total volume (r=0.65). A sample of 20 patients with the clinical diagnosis of Alzheimer's disease showed the correlation even higher (r=0.82). In patients with the Mild Cognitive Impairment (n=16), the correlation was 0.87. Using our protocol, clinicians can be informed about amygdalar size within a few minutes without the requirements of lengthy measurement.

Supported by grants IGA MZ 2006 NR8931‐4, and No. 0021620806 from the Ministry of Education of the Czech Republic

Grant Funding Source

IGA MZ CR NR8931‐4 ,
Functional evidence for polymerase switching in base excision repair by early zebrafish embryos
Authors: Fortier Sean , Wang Yi , Bennett Richard A.O. , Strauss Phyllis R
Published: June 2021

The base excision repair (BER) pathway recognizes and repairs most non‐bulky lesions, uracil and abasic (AP) sites in DNA. Since the pathway has never been investigated during embryogenesis, we characterized BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages and adults. Using a 45‐mer double stranded substrate with a U/G mispair at position 21, we showed that all extracts are capable of performing BER. Before 3 dpf aphidicolin‐sensitive polymerases perform most nucleotide insertion. After hatching, an aphidicolin‐resistant polymerase, probably DNA polymeraseß, becomes the primary polymerase. Previously we showed that when zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate equally as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early embryos and adults are the predominance of replicative polymerases and the presence of backup Mg2+‐dependent endonuclease activity in early stage embryos. Supported by funds from the National Institutes of Health 1R03HD045517, the G. Harold and Leila Y. Mathers Foundation, Aid for Cancer Research, and Northeastern University
Reconstitution of uracil DNA glycosylase initiated base excision repair in herpes simplex virus‐1
Authors: Boehmer Paul E , Bogani Federica , Chua Chian New
Published: June 2021

Herpes simplex virus‐1 is self‐sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV‐1 DNA polymerase (UL30) exhibits AP and 5′‐deoxyribose phosphate lyase activities that are integral to base excision repair (BER). We have mapped the lyase activity to the C‐terminal polymerase domain of UL30. This domain lacks polymerase activity but binds DNA. Candidate lysine residues in this fragment will be mutagenized to identify the lyase active site. We have also reconstituted a system with purified HSV‐1 and human proteins that performs all the steps of uracil DNA glycosylase initiated BER. In this system, nucleotide incorporation is dependent on the HSV‐1 uracil DNA glycosylase, AP endonuclease and UL30. Completion of BER is mediated by DNA ligase IIIα/XRCC1, which also confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV‐1 DNA polymerase processivity factor and prevents BER from occurring with heterologous DNA polymerases. These findings demonstrate that the HSV‐1 proteins in combination with cellular factors are capable of performing BER and have implications on viral genome maintenance during lytic replication and reactivation from latency.

This work was supported by grant GM 062643 from the NIH.
Transition mutations of Hirsutella sinensis genes and expressions of Paecilomyces hepiali genes during germination and early development of Cordyceps sinensis stroma
Authors: Yao Yisang , Zhou Yanjiao , Chen Wei , Zhu Jia‐Shi
Published: June 2021

We reported simultaneous detections of 2 live fungi, Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs), and their genes in Cordyceps sinensis (Cs) (J Mycol Res 5:214, 2007). With Cs maturation, Hs displayed a >50% decline of its competitive colony forming ability (p<0.001), and profiles of component proteins and small organic chemicals were altered. In this study with use of Ph (ITS 5‐179) and Hs (ITS 5‐162) specific probes, Southern blotting revealed large increases in expressions of Ph and Hs genes with Cs maturation. Two species of Hs‐related Cs genes (87% identity) were identified, consisting mainly of C‐T and G‐A mutations. In premature Cs, the GC genotype expressed only in caterpillar body, while mainly AT genotype in stroma. The Ph genes and the AT genotype Hs genes appear to express synchronously during germination and early development of Cs stroma. When Cs matures, the AT genotype starts to express in caterpillar and the GC genotype in stroma. It is assumed that essential enzymes needed for the transitions between GC and AT genotypes may be provided by co‐existing fungus Ph and play key roles in assisting the anamorph‐telemorph connection of Cs.
Dynamics differences of SAM‐I riboswitch aptamer between SAM bound and without SAM: insight into conformational rearrangement
Authors: Huang Wei , Aboul‐ela Fareed , Kim Joohyun , Jha Shantenu , Boyapati Vamsi
Published: June 2021

Increasing attention has focused on targeting RNAs for small molecule drug design. However, because RNAs are highly flexible, it is still not clear how the interactions between small molecules and RNAs are coupled with the conformational change of RNAs. A riboswitch is an autonomous cis‐regulatory element that controls gene expression in response to the signals from small molecule metabolites, such as amino acids, nucleotides, or enzyme cofactors etc. The SAM‐I riboswitch is one member of this regulatory family that can control the expression of biosynthetic proteins involved in sulfur metabolism through conformational change upon the binding of S‐adenosylmethione (SAM). Although the structure of SAM‐I riboswitch aptamer domain has been solved via X‐ray crystallography, it is just a static view of how SAM binds to SAM‐I riboswitch. In this study, molecular dynamics (MD) simulations are performed on SAM‐I riboswitch aptamer with SAM and without SAM. Principle component analysis (PCA) is applied to explore the global dynamics differences of SAM‐I riboswitch aptamer between SAM present and SAM absent. Additionally, other trajectory analyses are also used to probe the correlation between the local flexibility and the global motion. MD simulation results suggest that dynamics of the P4 helix is affected upon the binding of SAM. This change in dynamic behavior may be related to the conformational changes observed in the SAM‐I riboswitch.
Conformational Changes of Eukaryotic Chaperonin TRiC/CCT in the Nucleotide Cycle Revealed by CryoEM
Authors: Cong Yao , Schroeder Gunnar F. , Jakana Joanita , Reissmann Stefanie , Levitt Michael , Ludtke Steven J. , Frydman Judith , Chiu Wah
Published: June 2021

TRiC/CCT is a mammalian chaperonin made up of eight distinct polypeptides arranged in two rings. TRiC changes conformation in an ATP‐dependent manner which is critical to promote protein folding in the eukaryotic cell. Unlike prokaryotic chaperonins, substrate encapsulation within the central chamber of TRiC is triggered by ATP‐induced closure of a built‐in lid.

Our single particle cryo‐EM studies, including both reference‐free 2D analyses and 3D reconstructions, show the conformation variations of TRiC in different nucleotide states in the protein folding cycle. The observed changes are consistent with various biochemical evidences. Based on comparative modeling in combination with flexible fitting, pseudoatomic models have been built for each conformational state. Interestingly, in the most physiological related ATP hydrolysis transition (ADP‐AlFx) state, TRiC adopts an one ring open and one ring closed conformation. The closed ring is obviously expanded comparing to that in the closed (ATP‐AlFx) state. Notably, the inter‐domain motions in TRiC appear differently from those of prokaryotic chaperonins, despite their overall structural similarity.

This research is supported through NIH Roadmap Nanomedicine Initiative grant (PN2EY016525).
Mechanism in Conformational Change of Group II Chaperonin
Authors: Yohda Masafumi , Kanzaki Taro , Masuda Rie , Muhamad Sahlan , Oka Toshihiko
Published: June 2021

Previous studies have shown that ATP drives the conformational change of the group II chaperonin from the open‐lid, substrate binding conformation to the closed‐lid conformation. However, the detailed mechanism of the conformation change is still unknown. To elucidate this issue, we examined the effects of existence of conformational change‐deficient mutant (G65C), ATP hydrolysis‐deficient mutant (D64A/D393A) or helical protrusion‐deleted mutant (Δhelical, Δ245‐276) in the defined ordered fashion on the function of Thermococcus chaperonin complexes. Chaperonin complexes containing G65C or D64A/D393A alternately with wild type exhibit almost same refolding ability with the wild type complex. On the contrary, chaperonin complexes containing Δhelical could not fold denatured proteins. Interestingly, they also exhibited ATP‐dependent conformation change. Their conformation change is likely to be transient because they capture denatured protein even in the presence of ATP whereas addition of ATP impaired the ability of the wild type chaperonin to protect citrate synthase. These results suggest that ATP binding/hydrolysis induces the conformational change to the closed conformation, and its conformation is stabilized by the interaction between helical protrusions.
Is there a Way towards the Thermodynamics of Fibril Formation?
Authors: Buttstedt Anja , Sackewitz Mirko , Schmid Franz Xaver , Schwarz Elisabeth
Published: June 2021

The effect of the polypeptide environment on poly‐alanine induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling poly‐adenylation. Mutation‐caused extensions of the natural ten alanine sequence up to 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy. We explored the influence of fibril formation on the structure and function of a protein domain linked to the fibril‐forming part of PABPN1. A well‐characterized, stably folded one‐domain protein, cold shock protein B from Bacillus subtilis was fused either to the C‐terminus of the entire N‐terminal domain of PABPN1 or directly to a peptide consisting of ten alanine residues. The fusion protein of the N‐terminal domain and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the poly‐alanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid‐like fibrils and that the distance between this domain and the fibril plays an important role.
The effect of aging on the metabolic function and structure of mitochondria in Hamster oocytes
Authors: simsek‐duran fatma , li fang , ford wentia , swanson r. james , duran hakan e , jones howard w , castora frank j
Published: June 2021

In human oocytes there is a significant variation in the pregnancy potential. This low

proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. Hamster ova resemble human oocytes in size and fertility success rate and so serve as a model for understanding the human oocyte. ATP from the oocytes of young and aging hamsters was measured by a luciferase/luciferin reaction. On average, individual hamster oocytes from young animals (91 eggs from 6 animals) contain 144 ± 9.1 fmoles of ATP (0.16 uM). There is a 21% (P = 0.01) decrease in ATP (114 ± 10.1 fmoles, 0.14 uM in concentration) in old hamsters (88 eggs from 6 animals) compared

to young hamsters. RT‐ PCR was used to quantitate mtDNA in individual oocytes. This analysis indicated that there is also a decrease in mtDNA molecules per oocyte as the animal ages (11,466 ± 2,025 (young) vs 7,544 ± 2,507 (old), P= 0.07). The morphology of oocytes of young and old hamsters was examined by transmission electron microscopy. TEM indicated that mitochondrial morphology undergoes distinct changes as the hamster ages. In old hamsters, the cristae are decreased in number and length compared to those in young animals. In hamsters, the decrease in ATP levels seen in aged oocytes is correlated with a similar decrease in the number of mtDNA molecules. The consequence of these changes is a reduction of 25% in ATP concentration. Such a dramatic decrease in mitochondrial energy production could compromise normal oocyte biogenesis and contribute to the reduced pregnancy potential seen in aged mammalian oocytes.
Lipoic acid bioavailability in young and elderly adults
Authors: Butler Judy Ann , Hagen Tory M , Dixon Brian
Published: June 2021

(R)‐α ‐lipoic acid (LA) fed to old rats significantly increases low molecular weight antioxidants in multiple tissues and restores antioxidant defenses that otherwise decline with age. R‐LA improves resistance to toxicological insults, primarily by inducing a glutathione (GSH)‐mediated response and stress response pathways. A two‐phase pilot study seeks to determine whether the racemic (R,S‐) or the natural (R‐) form of LA is more effectively absorbed in young and elderly adults and whether LA improves indices known to be adversely affected by age. In the first phase, 10 young (18‐45) and 10 elderly (75 and older) adults receive a 500 mg tablet of either R‐ or R,S‐LA and blood samples are collected over the next 3 hours. After a washout period the study is repeated with the other form of LA. Plasma is isolated and concentrations of LA and GSH determined. In the second phase, 20 young and 20 elderly adults will be given the form of LA determined to be most bioavailable in elderly adults after completion of the first phase. Subjects will then take 500 mg LA or a placebo twice/day for one month followed by a washout period and crossover. Blood samples will be collected weekly to determine plasma GSH and LA concentrations, general blood chemistries and parameters of Phase II stress response pathways and GSH‐mediated responses will be monitored. Supported by Linus Pauling Institute through a gift from USANA Health Sciences, Inc.
High throughput resequencing to characterize age associated changes in nuclear factor erythroid 2 related factor 2 and the antioxidant response element interactions
Authors: Smith Eric Jonathan , Hagen Tory M
Published: June 2021

Interaction between Nuclear factor erythroid 2 related factor 2 (Nrf2,) a transcription factor, and all the classes of antioxidant response element (ARE) regulate important bioremediation of xenobiotics and reactive oxygen species. Nuclear levels of Nrf2 declines with age, as does its binding to a consensus ARE sequences. The glutamate‐cysteine ligase catalytic subunit (GCLC) gene is a quintessential Nrf2/ARE regulated gene that contains several AREs and codes for an enzyme essential for glutathione synthesis. We have previously shown that Nrf2 displays differential binding to the GCLC promoter at various AREs with age and that this change results in a decline in glutathione synthesis. Chromatin Immunoprecipitation (ChIP) coupled with qPCR has been a useful tool in defining the interactions between Nrf2 and the AREs in the GCLC promoter. Here ChIP has been coupled with high throughput resequencing (ChIP‐seq) to examine genome wide ARE and Nrf2 interaction in liver tissue isolated from young and old male F344 rats. Results show a dramatic change in the transcription factor binding profile between the two experimental groups.
Phosphate‐regulated phosphatases Phm8 and Sdt1 are essential for chronological lifespan in budding yeast
Authors: Bogan Katrina Leigh , Brenner Charles
Published: June 2021

Phosphate (Pi) is an essential and typically limiting nutrient for life. It is therefore critical that acquisition, uptake, storage, utilization and salvage of Pi be highly regulated. In S. cerevisiae, the PHO pathway regulates expression of genes that respond to low environmental Pi. The system includes a cyclin, cyclin‐dependent protein kinase and inhibitor complex, Pho80‐Pho85‐Pho81, the transcriptional activator, Pho4, and a variety of enzymes and targets that have been less well characterized. Here we identify two paralogous metabolite phosphatases, Phm8 and Sdt1, as targets and mediators of the PHO system. We show that Phm8 and Sdt1 are under negative regulation of the Pho80‐Pho85 complex, and are induced transcriptionally by Pho4 under Pi limiting conditions and at stationary phase. Chronological lifespan assays using knock‐out mutants reveal that Phm8 and Sdt1 are required in stationary phase for cells to remain viable and arrest properly in the G1 phase of the cell cycle. These findings define the roles of previously unknown effectors of the PHO system and implicate Pi metabolism as an important factor in chronological lifespan.
Characterization of peptide Methionine oxidation and Methionine sulfoxide reduction using Methionine‐rich proteins
Authors: Liang Xinwen , Zhang Yan , Le Dung Tien , Gladyshev Vadim N
Published: June 2021

Methionine (Met) is one of the amino acids particularly susceptible to oxidation. Methionine oxidation mediated by reactive oxygen species (ROS) may result in formation of methionine sulfoxide (MetO), alter protein structure/function, and accelerate age‐related diseases. However, MetO can be reduced back to Met by methionine sulfoxide reductases MsrA and MsrB. Therefore, MetO is a candidate for biomarker of oxidative stress and Msr deficiency. The conventional methods to monitor MetO formation and reduction rely on HPLC or mass spectrometry procedures. Here, we computationally identified six proteins rich in methionine residues (>20%), termed them Met‐rich proteins (MRPs), and used them to examine Met oxidation and repair by in‐gel and immunoblot assays. Met oxidation and MetO reduction could be easily monitored by the mobility shift of these proteins on SDS‐PAGE gels, and by immunoblot assays with antibodies specific for the oxidized forms of MRPs. The oxidation of MRPs with hydrogen peroxide was more selective to Met than that with hypochlorite or other oxidants. All MetO residues in MRPs could by reduced by MsrA and MsrB either in DTT or Trx dependent assays. Our research suggests that MRPs may be a useful and convenient tool to characterize Met oxidation, MetO reduction and Msr activity.
Aortic endothelial cells transport more glucose than pulmonary endothelial cells in vitro
Authors: Viator Ryan Joseph , Fouty Brian William
Published: June 2021

Diabetes is characterized by hyperglycemia resulting from insulin resistance or deficiency. Increases in intracellular glucose can lead to endothelial dysfunction. While systemic vascular disease is a major cause of morbidity and mortality in diabetics, pulmonary vascular disease is less clinically apparent. Since both circulations are exposed to similar circulating glucose and insulin concentrations, we hypothesize that increased glucose loading of systemic endothelial cells might explain this difference. Using bovine aortic and pulmonary artery endothelial cells from the same animal (AECs or PAECs, respectively), we found that AECs transported 5‐fold more glucose than PAECs. Because shear stress is different between the two vascular beds, we examined the effect of shear stress on glucose transport. Flow adapted cells decreased radiolabelled glucose uptake but remained higher in AECs. Glucose enters endothelial cells primarily through the glucose transporter, Glut‐1. To determine if an increase in Glut‐1 expression might explain the difference in glucose uptake, we compared cells using Western Blot Analysis. AECs expressed more Glut‐1 than the PAECs. The increased glucose loading of AECs compared to PAECs may be important in determining the different effects of diabetes in the systemic and pulmonary circulations. This research is supported by the NIH grants R01HL070273 and T32HL076125.
RGS::GFP expression in endocrine pancreas during development and regeneration
Authors: Wilkie Tom , Villasenor Alethia , Wang Zhao , Hatten Mary E , Scherer Philipp , Cleaver Ondine
Published: June 2021

Regulation of G protein signaling provides a promising approach for managing beta cell replication and neogenesis. RGS proteins are intracellular GTPase activating proteins (GAPs) that terminate ligand‐activated G protein coupled receptor (GPCR) signaling. We characterized expression of a GFP‐reporter gene integrated into two distinct RGS genes, Rgs8 and Rgs16, in embryonic and adult pancreas. Rgs16::GFP and Rgs8::GFP were expressed in pancreatic endocrine progenitor cells in embryos, neonates and weanlings but their expression was normally quiescent in adulthood. Rgs16 expression was reactivated in four different models of beta cell expansion: i) weanlings, ii) mid gestation pregnant females, iii) during islet regeneration in PANIC‐ATTAC mice, a model of type 1 diabetes and iv) in islets of obese, hyperglycemic ob/ob mice, a model of type 2 diabetes. Our results suggest that Rgs16 and Rgs8 control GPCR signaling during islet progenitor cell activation, differentiation, and beta cell expansion in embryos and metabolically stressed adults.

Research support: NIGMS & Welch Foundation.
Differential protein expression between normal and myxomatous canine mitral valves
Authors: Lacerda Carla MR , Orton Christopher
Published: June 2021

Each year, valvular heart disease accounts for over 20,000 deaths in the U.S. Myxomatous valve disease (MVD) is the most common heart valve disease in humans and dogs. MVD is pathologically identical in these species, and its pathogenesis is poorly understood. The study objectives were to 1) develop proteomic methodology suitable for analysis of extracellular matrix‐rich heart valve tissues, and 2) survey over‐ and under‐expressed proteins that could provide mechanistic clues into the pathogenesis of MVD.

METHODS

Normal, early‐stage and late‐stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis of extracted proteins with iTRAQ labeling to determine differential protein quantification was employed. Proteins were classified by function and clustered according to differential expression patterns.

RESULTS

More than 300 proteins, with 115 of those being differentially expressed, were identified. Sample clustering showed that early‐ and late‐stage valves were closely‐related, when compared to normal valves.

CONCLUSIONS

Shotgun proteomic analysis of matrix‐rich canine heart valves is feasible, and should be applicable to human heart valves as well. Differential protein expression provides a basis for future investigations into the pathogenesis of canine and human MVD. Research supported by Heart to Heart foundation.
Proteomic analysis of plasma protein in β‐thalassemia/HbE patients treated with curcumin
Authors: Kalpravidh Ruchaneekorn , Weeraphan Churat , Srisomsap Chantragan , Siritanaratkul Noppadol , Svasti M.R. Jisnuson
Published: June 2021

Thalassemia is a group of genetic disorders resulting from different mutations in the globin gene clusters and leading to an imbalance in globin synthesis. Unpaired globin chains are less stable and are susceptible to oxidation. Patients with beta‐thalassemia/HbE (β‐thal/HbE) are prone to increase oxidative stress as indicated by increased lipid peroxidation and protein oxidation. Curcumin, a strong antioxidant and iron chelator, has been shown to decrease oxidative stress in β‐thal/HbE patients. In this study, the effect of curcumin on plasma protein expression was investigated using proteomic analysis in untreated patients as compared to normal subjects and in patients before and after supplementing with 500 mg curcumin daily for 12 months. Plasma proteins involved in blood coagulation and hemostasis e.g. prothrombin, fibrinogen, hemopexin, and haptoglobin were significantly decreased in patients, particularly in splenectomized patients. The administration of curcumin led to significant increase of prothrombin, fibrinogen, and haptoglobin. Our results provide strong evidence for the existence of a chronic hypercoagulable state in thalassemia and the patients responded to curcumin supplement as shown by a rise in the level of procoagulant proteins. Therefore, the administration of curcumin could be beneficial for thalassemic patients as adjunct therapy to decrease hypercoagulable state.
Proteomics analysis of autophagic cells under starvation
Authors: Kang Jeong Han , Ding Wen‐Xing , Liao Yong , Yin Xiao‐Ming
Published: June 2021

Autophagy is involved in the bulk degradation of long‐lived cytosolic proteins and subcellular organelles, and is important for the survival of cells during starvation and stress. The underlying mechanisms are still unclear. A comparative proteomics approach, combining two‐dimensional gel‐electrophoresis with matrix‐assisted laser desorption/ionization time‐of flight mass spectrometry, was used to identify proteins whose expression is changed under amino acid starvation‐induced autophagy cells. We anticipated that this study might provide clues on protein regulations that may be important for autophagy process. In total, the expression levels of 58 protein spots changed significantly between the starved cells and control cells. The large differences in protein expressions were observed for enzymes involved in intermediate metabolism, structure proteins and proteins associated with subcellular organelles. The expression of some proteins was validated by RT‐PCR, Immunoblotting, immunofluorescent microscopy and functional activity assays. The data described here will contribute to establishing a functional profile of proteome in autophagy‐induced cells. Furthermore, the proteins identified in this study may be attractive candidates as biomarkers for any pathogenesis processes involving autophagy.
The 32 Codon World of A. dehalogenans
Authors: Duax William L , Huether Robert , Pletnev Vladimir , Kirsch Tyler , Hogan Dana , Teysir Jimmitti , Benatovich Sam , Bednarz Jane , Dziak David , McCauley Maddy
Published: June 2021

The genome of the soil bacteria Anaeromyxobacter dehalogenans (Adhal) is reported to have 4346 coding sequences composed of 1,521,374 codons, a GC content of 74.82% and GC occupancy of the third base position of 97.07%. Over 50% of the proteins in Adhal are annotated as having uncertain or unknown function. Over 1000 have two or less Met residues and Val and Leu residues are often identified as start codes. Examination of the sequences indicates that over 80% of them have 3 open reading frames. The presence of multiple open reading frames and ambiguity of start and stop code identification has resulted in misidentification of hundreds of nonsense sequences as hypothetical proteins. Significant differences in codon use between functionally characterized and hypothetical proteins, errors in coding frame selection, and in start and stop code identifications indicate that most (and possibly all) of the sixteen codons that have A or T bases in the first and third position are nonsense codes (de facto stops). These patterns present in many bacterial genomes suggest that the modern genetic code evolved from a coding system in which just the 32 codons that end in G or C define all 20 amino acids. The DNA in which this code evolved was stabilized by the presence of three hydrogen bonds linking the GC pairs present in every third base pair of the DNA encoding proteins.
A mutation in a COG4 homologue affects polarity establishment in the fungus Aspergillus nidulans
Authors: Gremillion Sara , Camara Kaddy , Mercer Sarah , Samuels Felicia , Hill Terry , Loprete Darlene
Published: June 2021

We have identified a mutation in the fungus Aspergillus nidulans, designated swoP (for swollen cell phenotype), causing a temperature‐sensitive morphological defect during spore germination and polarized, hyphal growth. At 42C conidia swell to approximately 1.5 times the normal diameter and establish multiple points of polarity, which grow isotropically before arrestGrowth at 30C is essentially normal, though hyphae are slightly wider than wild type and exhibit a minor "steering" defect. Cells grown at 42C for up to 18 hours recover normal hyphal morphology upon transfer to 30C. In hyphae transferred from 30C to 42C, both terminal and sub‐apical compartments swell irregularly and may produce multiple buds. The growth defect of swoP is complemented by the wild type allele of AN7462, which shows strong sequence homology to COG4, a component of the Golgi tethering complex associated with retrograde transport of vesicles. Sequencing of the COG4 allele of the swoP mutant reveals a mutation at base pair 2672, introducing a stop codon at amino acid 780. Meiotic mapping produces a ca. 12% recombination frequency between the swoP and AcuK (acetate) loci, which is consistent with the chromosomal location of AN7462. Through high copy gene expression, functional connections were found between the COG4 homologue to other COG proteins, thus providing further support of COG4 involvement in polarity maintenance.
The Coffin‐Lowry syndrome‐associated protein RSK2 is implicated in calcium‐regulated exocytosis through the regulation of PLD1
Authors: Vitale Nicolas , Zeniou‐Meyer Maria , Béglé Aurélie , Bader Marie‐France
Published: June 2021

Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins such as SNAREs, and also specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone‐shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)‐dependent phosphorylation. RSK2 is activated by a high K+induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knock down of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino‐terminal PX‐domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD‐related defects in neuronal and endocrine activities could contribute to the effect observed after the loss of function mutations in Rsk2 that leads to Coffin‐Lowry syndrome (CLS), an X‐linked form of growth and mental retardation.
Improving In Vivo Retention of Bioscavengers In Blood
Authors: Calderon Frances Suiyin , Campbell Amy J. , Moorad‐Doctor Deborah , Garcia Greg E.
Published: June 2021

We are developing technologies to increase the in vivo retention times of bioscavengers by attaching them to red blood cells (RBCs) using cell‐penetrating peptides (CPPs). CPPs are short peptides of 5‐40 amino acids, with the ability to translocate inside the cells by different mechanisms including direct physical transfer through the lipid bilayer. CPPs can either anchor the bioscavnegers to the cell surface, or internalize them by associated transport. Our results show that conjugation of the bioscavenger butyrylcholinesterase (BChE) to CPPs differently affected the enzymatic activity. Conjugation of Buforin I impaired the BChE activity by 50%. In contrast, the HIV Tat‐protein transduction domain (Tat) reduced the BChE activity by 11% in comparison to the unconjugated BChE. Tat was also found to significantly increase the association of BChE activity to RBC. Association of Tat to RBCs was also monitored by confocal microscopy using fluorescent microspheres. In addition, we are using artificial liposomes mimicking the RBC membrane composition as a further way to test CPP association to biological membranes. We are also investigating the capability of other RBC‐interacting peptides to associate bioscavengers. We expect that such technologies increase the effectiveness of protein therapeutics, or prophylactics, in blood borne treatments.

Supported by DTRA#1.D0011_08_WR_C.
The Structures of COPII Coats and Cages
Authors: Stagg Scott Malone , LaPointe Paul , Razvi Abbas , Gurkan Cemal , Potter Clinton S , Carragher Bridget , Balch William E
Published: June 2021

Coat protein complex II (COPII) vesicles are responsible for packaging and transporting over 10,000 different cargo molecules (about one‐third of the eukaryotic genome) of widely varying sizes and shapes from the endoplasmic reticulum (ER) to downstream compartments of the secretory pathway. Proteins involved in generating COPII vesicles include the GTPase Sar1, the cargo adaptor Sec23/24, and the cage scaffold Sec13/31. We previously showed that Sec13/31 can self‐assemble into a novel cuboctahedron cage, leading us to suggest that Sec13/31 may form cages of increasing size based on the simple rule that the geometry of the cage is dictated by four Sec13/31 heterotetramers combining to form a vertex. We have now solved the structure of an icosidodecahedral COPII coat assembled from both Sec13/31 and Sec23/24 by cryo‐electron microscopy (cryoEM) and single particle reconstruction. This new structure reveals for the first time the structure of the adaptor layer and reveals possible mechanisms for the coordination of adaptor interaction with cage formation and for the collection of cargo of varying size. We fit a crystal structure of the Sec13/31 heterotetramer into the cuboctahedron and icosidodecahedron structures, and this lead us to propose a molecular mechanism by which the COPII system can accommodate cargo of diverse sizes and shapes.
Biased agonism reveals new G protein‐independent AT1a receptor signals
Authors: Kendall Ryan Thomas , Lee Mi‐Hye , El‐Shewy Hesham M. , Janech Michael G. , Luttrell Deirdre K. , Luttrell Louis M.
Published: June 2021

The angiotensin II analog [Sar1, Ile4, Ile8]AngII (SII) is a biased AT1a receptor agonist that stimulates receptor phosphorylation, βarrestin recruitment, internalization and βarrestin‐dependent ERK1/2 activation‐without activating heterotrimeric G proteins. To determine the scope of G protein‐independent AT1a receptor signaling, we performed a phosphoproteomic analysis of SII‐induced signaling in HEK cells stably expressing AT1a receptors. A total of 26 differentially abundant proteins were analyzed by phosphostaining 2D gels and identified by peptide mass fingerprinting. Phosphorylation of two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A), prostaglandin E synthase 3 (PGES3) and casein kinase (CK2β) was confirmed by immunoprecipitation and immunoblotting with phospho‐specific probes. Functionally, SII stimulation produced rapid and transient inhibition of PP2A activity in HEK cells and stimulated PGE2 synthesis in primary vascular smooth muscle. These findings suggest that AT1a receptors regulate a robust G protein‐independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production, and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.

This work was supported by NIH grants HL07260 (R.T.K.) and DK55524 (L.M.L.) and a Department of Veterans Affairs REAP Awards.
Neuropeptide Y (NPY) forms stable complexes with the Y1 receptor and G‐protein alpha subunits, reducing the transduction and internalization of the receptor
Authors: Parker Steven L , Parker Michael S , Sah Renu , Balasubramaniam Ambikaipakan , Sallee Floyd R
Published: June 2021

Neuropeptide Y (NPY) and peptide YY (PYY) bind to similar numbers of high‐affinity sites in both native and clonal expressions of the NPY Y1 receptor. However, NPY binds to a much larger number of sites poorly competed by NPY or PYY, or by Y1‐selective agonist BIBP‐3226. This 'non‐specific' binding is much larger with the brain Y1 receptor, apparently due to high G‐protein content. The sites are associated largely with the pertussis toxin‐persisting fraction of ~90 kDa complexes of the monomeric receptor and G‐protein alpha subunits, and are not apparent in ~180 kDa heteropentamers of the Y1 receptor dimer and non‐activated G‐protein heterotrimers. High concentrations of NPY (but not of PYY) block the surface Y1 sites and internalization in CHO cells. Inhibition of adenylate cyclase by NPY in Y1‐CHO cells is significantly less than that by PYY. These findings indicate that Y1 receptor‐attached NPY could also bind to G‐protein alpha subunits associated with the receptor, inducing lower activation, transduction, and cycling. This could be important in the regulation of activity and in conservation of both the Y1 receptor and G‐proteins in NPY‐rich environments, e.g. in the forebrain.
Ca2+‐sensitive redistribution of calmodulin and neurogranin in CA1 pyramidal neurons of mouse hippocampus
Authors: Huang Kuo‐Ping , Huang Freesia L.
Published: June 2021

Our previous studies with neroganin (Ng) knockout mice suggested that Ng was important for increasing Ca2+‐transients and activation of Ca2+/CaM‐dependent enzymes that were essential for the enhancement of synaptic plasticity and long‐term memory formation. In vitro, Ng has been shown to be an apo‐CaM‐binding protein; however, their interactions in situ haven't been established. Fluorescence immunochemical staining of hippocampal slices bathed in Ca2+‐containing artificial cerebral spinal fluid (ACSF) revealed that CaM and Ng were co‐localized in the principle neurons of the hippocampus. The concentration of CaM in the nucleus was much higher than that of cytoplasm and dendrites, whereas Ng was more evenly distributed throughout the various cellular compartments. Changing the bathing fluid from the Ca2+‐containing to the Ca2+‐free (but containing 2.5 mM EGTA) ACSF resulted in a redistribution of CaM and Ng from soma to dendrites. Surprisingly, this redistribution occurred mainly in the CA1 and was not evident in the other hippocampal subfields, including CA2, CA3, CA4, and dentate gyrus. Re‐perfusion with the Ca2+‐containing ACSF caused a complete relocation of these two proteins to their original states. These results illustrate that the Ca2+‐sensitive coordinated movement of Ng and CaM may be essential for the enhancement of synaptic plasticity in the CA1 pyramidal neurons. (Supported by NICHD, NIH)
Structural interactions of calmodulin with peptide from calmodulin binding regions of various enzymes
Authors: Duangkham Yay , Spratt Donald , Dieckmann Thorsten , Guillemette J. Guy
Published: June 2021

Calmodulin (CaM) is an important calcium (Ca2+) sensor in the cell and is able to activate over three hundred proteins to initiate cellular activities. CaM is a highly dynamic protein with a flexible central linker region joining the N‐terminal and C‐terminal domains. In each domain are two Ca2+ binding sites that cause structural changes to CaM when Ca2+ binds. These changes include the CaM central linker elongating and methionine rich hydrophobic patches being exposed, allowing for CaM to bind to the large population of target proteins. The structural interaction of CaM to short peptides from the CaM binding region of various target enzymes were investigated, including the nitric oxide synthase (NOS) enzyme.
1α, 25‐Dihydroxyvitamin D Regulates Hypoxia‐Inducible Factor‐1α in Breast Epithelial Cells
Authors: Jiang Yan , Harpenau Erin M. , Teegarden Dorothy
Published: June 2021

The purpose of this study was to determine the impact of 1α, 25 dihydroxyvitamin D (1,25(OH)2D) on hypoxia‐inducible factor 1α (HIF‐1α) expression, an important angiogenic transcription factor, in MCF10A and oncogene (H‐ras and ErbB2) transfected MCF10A breast epithelial cells which are models of cancer progression. Treatment with 1,25(OH)2D (10 nM) increased both mRNA (64% + 19 greater than vehicle, p=0.01) and protein (370% + 41 greater than vehicle, p<0.0001) of HIF‐1α in MCF10A cells in 12 hours. After 24 hrs, HIF‐1α mRNA expression returned to baseline, but HIF‐1α protein level remained higher (590%+150 greater than vehicle, p=0.02). Treatment with a transcriptional inhibitor, actinomycin D, prevented the 1,25(OH)2D induction of HIF‐1α protein expression in MCF10A cells. In contrast, treatment with 1,25(OH)2D (24 hr) did not change HIF‐1α protein expression in H‐ras transfected or ErbB2‐transfected MCF10A cells. However, mRNA level of HIF‐1α was decreased (23%+4.6 less than vehicle, p=0.008) in the H‐ras transfected cells, but was not different with 1,25(OH)2D (24 hr) treatment in ErbB2‐transfected cells compared to vehicle. These results suggest that 1,25(OH)2D induction of HIF‐1α expression is through transcriptional regulation in MCF10A breast epithelial cells and the effect may be modulated in the presence of H‐ras or ErbB2 oncogenes. Supported by NIH DK069965