bluuebunny/crossref_metadata_2025 huggingface dataset:
NMDA channel activation regulates Ca2+ permeation and gene transcription during histamine‐stimulated gastric acid secretion (904.5)
Authors: Seo Ji Hye , Itagaki Kiyoshi , Wang Yaoyu , Hagen Susan
Published: June 2021

Background: Histamine‐stimulated gastric acid secretion is accompanied by oscillations in intracellular Ca2+. The receptor mediating these oscillations and the function of this Ca2+ signal are unknown. We aimed to test the hypothesis that histamine stimulation activates N‐methyl‐D‐aspartate (NMDA) channel‐mediated Ca2+ influx and gene transcription in parietal cells. Methods: Cultured rat parietal cells were stimulated with histamine plus IBMX with or without NMDA channel antagonists. Intracellular Ca2+ was visualized by microscopy using fura‐2 or fluo‐3. Western blots and RNA‐Seq were used to evaluate CREB/ATF‐1 activation and gene transcription, respectively. Results: Histamine stimulation induced Ca2+ oscillations/permeation that were blocked by NMDA channel antagonists. CREB and ATF‐1 were activated by histamine stimulation and blocked by NMDA channel antagonists. Transcriptome analysis identified numerous up‐ and down‐regulated genes linked to histamine stimulation with a subset affected by NMDA channel blockade. The top network function attributed to NMDA channel‐mediated genes was cell death and survival. Most of the highly up‐regulated genes were CREB target genes. Conclusions: Histamine‐stimulated Ca2+ oscillations are regulated by NMDA channels. Our data show that this pool of intracellular Ca2+ transcriptionally regulates cell survival‐associated genes in parietal cells.
Ferristatin II induces hepatic hepcidin expression and Stat3 phosphorylation (907.3)
Authors: Alkhateeb Ahmed , Buckett Peter , Kim Jonghan , Byrne Shaina , Wessling‐Resnick Marianne
Published: June 2021

Administration of Ferristatin II (FS2) to rats is associated with decreased serum iron, reduced transferrin saturation and increased hepatic hepcidin expression without changes in liver non‐heme iron levels. To better understand the mechanisms underlying hepcidin induction by FS2, its effects on ER stress were examined. XBP1 splicing, a marker of ER stress, was not altered in livers from treated rats. Likewise, levels of the ER stress protein Grp94 were unchanged in FS2‐treated Hep3B cells. SMAD signaling, a second regulatory pathway, did not appear to be activated since transcript levels of downstream targets SMAD7, BMP6 and Id1 were all reduced in livers of FS2‐treated rats. However, mRNA levels of alpha‐2‐microglobulin, alpha‐1‐acid glycoprotein and C‐reactive protein were up‐regulated. Western blot experiments confirmed that FS2 treatment induced phosphorylation of hepatic Stat3, a known mediator of the acute phase response and a regulator of hepcidin expression. Accordingly, FS2 induced Stat3 phosphorylation in HepG2 cells in vitro. These results suggest that FS2 induces hepatic hepcidin expression in a Stat3‐dependent manner. The pharmacological induction of hepcidin by ferristatin II holds clinical potential to decrease iron absorption in iron overload diseases.

Grant Funding Source: Supported by NIH NIDDK (DK064750)
Inflammation reduces the activity of the NaHCO3 cotransporter, NBCe1, in the surface cells of the proximal colon of IL10 knockout mice (908.1)
Authors: Fan Shjun , Schultz Michael , Butt Grant
Published: June 2021

The impact of inflammation on intestinal secretion is contentious. Here the effect of inflammation on activity of the NaHCO3 cotransporter, NBCe1, in the proximal colon was investigated. Interleukin10‐knockout (IL10‐/‐) mice infected with Helicobacter typhlonius, which develop colitis, were used as a model of inflammation, and uninfected, asymptomatic IL10‐/‐ mice served as controls. NBCe1 activity was measured as the 5‐(N‐ethyl‐N‐isopropyl) amiloride‐insensitive (EIPA, 20µM), 4,4'‐diisothiocyano‐2,2'‐stilbenedisulfonic acid‐sensitive (DIDS, 500µM), rate of intracellular pH recovery and acid extrusion (JH+) following acid loading of isolated crypts, while NBCe1 expression was measured by Western blotting and immunohistochemistry. In control tissues NBCe1 immunoreactivity was restricted to the surface cells and upper third of the crypts. Consistent with this there was little evidence of NBCe1 activity in the lower half of the crypts, with or without forskolin (10µM) stimulation. In contrast, in the surface cells, while NBCe1 was inactive under basal conditions (JH+EIPA=16.4±1.8mM.min‐1; JH+EIPA+DIDS=18.7±1.8 mM.min‐1), its activity was stimulated by forskolin (after forskolin JH+EIPA=24.5±2.7 mM.min‐1 and JH+EIPA+DIDS=13.6±1.1mM.min‐1). In inflamed tissues, total NBCe1 expression was reduced (P<0.001, n=6), there was no NBCe1 immunoreactivity in the surface cells and upper crypts, and forskolin did not stimulate NBCe1 activity in the surface cells. This suggests inflammation reduces colonic HCO3‐ secretion, which may affect luminal pH regulation and growth of luminal bacteria, possibly contributing to the dysbiosis seen in patients with inflammatory bowel disease, as well as modifying mucus hydration.

Grant Funding Source: Supported by The Otago Medical Research Foundation
Bile acid chenodeoxycholate activation of Cl‐ transport involves epidermal growth factor receptor signal transduction in human colonic T84 cells (908.4)
Authors: Domingue Jada , Ao Mei , Sarathy Jayashree , Alrefai Waddah , Rao Mrinalini
Published: June 2021

A role for EGFR signaling in BA action has been well described in colon cancer, but not as well in BA induced Cl‐ secretion. Our preliminary report (EB LB 10695, ‘13) showed that the EGFR inhibitor, AG1478, reduced CDCA (500 µM) mediated Cl‐ secretion by 69%, measured as short circuit current (Isc, µA.cm‐2), in T84 cells. Here we aim to further elucidate this mechanism. To determine if BA action involves matrix metalloproteinases (MMP), CDCA (500 µM) was added to T84 cells ± batimastat (BAT, 20 µM, 30’). CDCA itself increases Isc in 2‐3’ and the effect is sustained for > 20’. BAT inhibited the sustained phase (∆Isc: CDCA: 11.6±1.4; +BAT: 3.9±2.6, n=3). Interestingly the PKA inhibitor H89 has a similar effect (∆Isc: CDCA: 21±4.6; +H89 (30 µM): 1±0.3, n=5), suggesting that EGF and cAMP are involved in sustaining CDCA‐stimulated secretion. The distal steps of EGFR action do not involve Src or MEK as their respective inhibitors PP2 (10 µM, n=3), and PD98059 (20 µM, n=3) did not alter CDCA‐induced Isc. Immunoblotting showed CDCA (5’) increased EGFR (n=1), p38 (n=3), and ERK 1/2 (n=3) phosphorylation. Based on this data we postulate that CDCA stimulates Cl‐ secretion via MMP‐EGFR‐p38 signaling, but not via Src or the MEK‐ERK pathways. Together with our data (AJP 305:C447‐456, ‘13) that CDCA acts via PKA to activate CFTR, we summarize that CDCA evokes multiple signaling cascades and their cross talk needs further elucidation.

Grant Funding Source: Supported by NIH and VA
The effects of insulin on gonadotropin‐releasing hormone secretion (911.1)
Authors: Herrera Danny , DiVall Sara , Wolfe Andrew
Published: June 2021

The purpose of this study was to determine the role of insulin signaling in gonadotropin‐releasing hormone (GnRH) pulse generation. The Cre‐lox system was used to generate mice without insulin receptors in GnRH neurons (GnRH Cre+ IR fl/fl; GnRHIRKO). Hypothalami from female GnRHIRKO or littermate controls with intact insulin receptors in GnRH neurons (GnRH Cre‐), in the lean or diet induced obesity (DIO) state were incubated in static culture. Media collected every seven minutes was subjected to radioimmunoassay for GnRH. The pulse pattern in the control DIO state was visually less regular than other states. GnRH pulse amplitude was not different between GnRHIRKO or control mice in the lean or DIO state. Pulse frequency was trended lower in the control DIO state than in lean controls or GnRHIRKO lean or DIO hypothalami. However, this result was not statistically significant. Pulse interval was also longer in the control DIO state compared to the control lean or GnRHIRKO hypothalami. Insulin signaling in the GnRH neuron may play a role in GnRH pulse generation in the DIO but not the lean state.

Grant Funding Source: Supported by: NIH K08HD056139 and APS Undergraduate Summer Research Fellowship
Endocrine disruption in endangered Oregon spotted frogs (Rana pretiosa) (911.9)
Authors: Griffing Aaron , Sessions Stanley
Published: June 2021

The Oregon spotted frog (Rana pretiosa) is an endangered species residing in the Pacific Northwest whose dwindling numbers having caught the attention of biologists and environmental scientists. We tested the idea that R. pretiosa populations may be declining due to disruption of sex determination via endocrine disruption and/or parasite infection. Newly metamorphosed specimens were collected from localities in Oregon and their gonads were examined using paraffin histology. The sex ratio was 41% males and 59% females. This was not statistically significant. We found that 46% of the male frogs had oocytes developing in their testes. We also found that all specimens had numerous trematode cysts in their kidneys. These results from wild‐caught frogs were compared to an analysis of captive‐raised Wood Frogs (Rana sylvatica) from New York.

Grant Funding Source: Supported by the Oregon Community Foundation and its grant
The anthropometric parameters of organic evolution at children of junior school age in Astana (913.1)
Authors: Suleimenova F.M. , Aubakirov A.B. , Maul Y.Y. , Adaibayev T.A. , Dosmambetova K.K.
Published: June 2021

To research some anatomical and anthropometrical parameters of physical development at children ‐ Kazakhs (from 7 till 11 years), living in Astana city. Material and methods: 1102 children (547 boys and 555 girls)were observed. In research are used the anthropometrical methods. The results:the research showed that the sudden change inat the boys growingis marked in 10‐year's age, and at the girls the peak of this parameter is observein 9 ‐yearsold. At the girls the weight parameters are increaseinsignificantlyin 9‐ and 11‐year's age, but in 11 years old its conceded to the weight of the boys (on the average at 1,57kg). The parameters oflung vital capacity at younger school agechildren accordwith usual age changes without sudden changeand rises, that is are increased gradually. The plantometrical research have shown that minimal percent of flat‐footedness is marked at children in 7 years old. However to 11 years old this parameter is increased at the boys up to 3,7%, and at the girls up to 4,08% of cases. At the 9‐year's girls the biological age advance the chronological in 52,0+0,1% of cases, at the boys reaches a maximum in 10‐year's age and makes 33,08% of cases. The maximal parameter of lagging in biological development both at the boys and at the girls is determined in 11 years. The maximal sudden changeof anthropometrical parameters is marked at the girls in 9‐year's age, practically in 100% of cases.
Bite force patterns along the tooth row in anthropoid primates (918.15)
Authors: Lucas Lynn
Published: June 2021

Models of biting mechanics have formed the basis for understanding how bite force is distributed along the tooth row, and play a major role in the interpretation of variation in facial morphology in both extant and extinct primates. However, the pattern of predicted force distribution differs depending on the model used. While simple lever models predict a steady increase in bite force as the bite point moves distally along the tooth row (e.g., Gysi, 1921; Smith, 1978), constrained lever models that address the weakness of the temporomandibular joint to tensile loading‐which may impact biting on posterior teeth in some mammals‐predict that forces will plateau (Greaves, 1978) or decrease (Spencer, 1999) with more posterior bites. Experimental studies have variously supported the predictions of both simple and constrained lever models. To determine whether biomechanical models would predict similar bite force patterns in primates with varying skull morphologies, I measured the facial skeletons of over 200 anthropoid individuals and estimated bite force curves according to Spencer’s (1999) model of feeding mechanics. Results indicate that bite forces both within and among taxa may follow any of the patterns predicted by models of bite force mechanics, depending on the facial morphology of the individual specimen, suggesting that models of bite mechanics alone cannot predict bite force patterns along the tooth row.
miRNAs as the molecular clock regulating mandibular osteoinduction (921.5)
Authors: Roberts Ryan , Merrill Amy
Published: June 2021

Precise timing of cranial neural crest cell (CNCC) differentiation into bone is essential for proper formation of the craniofacial bones. Delayed osteoinduction leads to micrognathia, whereas premature osteoinduction causes craniosynostosis. To understand the etiology of these birth defects, we must uncover the molecular clock that regulates the onset of osteogenesis. CNCC autonomously control the timing of their differentiation into osteoblasts by regulating expression of Bone Morphogenetic Protein (BMP), yet how CNCC establish the timing of BMP expression remains unclear. Good candidates for intrinsic temporal control of gene expression are miRNAs, small non‐coding RNAs that target mRNAs for degradation. To identify specific miRNAs involved in mandibular osteogenesis, we performed microarray analysis on chick mandibles during the stages of osteoinduction. We identified and validated temporal expression changes in miRNAs whose targets include BMP signaling pathway members that are known to be involved in bone formation. For example, miR‐19b, which is upregulated at the start of osteogenesis, targets repressors of BMP signaling including Smurf1. Conversely, let‐7a and let‐7c, which are upregulated at the close of osteoinduction, target the BMP receptor AcvR‐2B. These results suggest that miRNAs intrinsically control the timing of CNCC osteoinduction by regulating the period of active BMP signaling.

Grant Funding Source: NIH 5P30DE020750‐02
Molecular mechanisms that mediate mechanosensation during secondary chondrogenesis (921.9)
Authors: Woronowicz Katherine , Hague Molly , Schneider Richard
Published: June 2021

The ability of cells to detect mechanical forces plays an essential role in generating the proper form and function of the craniofacial musculoskeletal system. One skeletal tissue that remains highly responsive to mechanical force is secondary cartilage, which develops along the surface of bone. Without appropriate mechanical stimulation, such as the forces that arise through motility of the embryo, secondary cartilage does not form, or degenerates. To elucidate how secondary cartilage is induced and maintained by mechanical force, we take advantage of the fact that secondary cartilage is closely associated with the specialized feeding of some species of birds. Duck utilize their jaws as a means to filter heavy sediment while chick peck at seed. Embryonic motility in duck induces secondary cartilage within the jaw adductor enthesis, creating a robust interface between tendon and bone on the coronoid process, and ultimately transducing the powerful muscle contractions needed to lift the jaw. In contrast, secondary cartilage is absent on the chick coronoid process. Our published and preliminary data lead us to hypothesize that species‐specific jaw anatomy generates dissimilar mechanical forces that differentially activate mechanosensitive Fibroblast Growth Factor and Calcium signaling pathways in duck versus chick, and instruct the duck jaw adductor enthesis to produce secondary cartilage. We employ gain‐ and loss‐of‐function approaches to modulate secondary chondrogenesis, as well as chick‐duck chimeras to test if chick cells can form secondary cartilage in a duck host environment. This research has the potential to translate into regenerative therapies to supplement surgical repair of torn and damaged muscle insertions.

Grant Funding Source: Supported by NIH 5R01DE016402‐08
Expression and immunohistochemical localization of the Gβγ‐activated and calcineurin‐inhibited adenylyl cyclase isoforms in rat articular chondrocytes (922.9)
Authors: Memon Ismail , Khan Khalid , Siddiqui Sammer , Perveen Siddiqa , Ishaq Muhammad
Published: June 2021

Aims: Cross‐talk between signal transduction pathways is essential to maintain normal homeostasis. cAMP mediated pathway is archetypical in mammalian cells and adenylyl cyclase (AC) is the effector enzyme that converts ATP into cAMP. We believe that AC/cAMP mediated pathway is involved in homeostasis of the articular cartilage. In this study we report the expression of Gβγ‐activated AC isoforms 2, 4, and 7 and calcineurin‐inhibited AC isoform 9 in rat articular chondrocytes.

Main methods: Fresh slices of articular cartilage were taken from various synovial joints of rat and the expression of AC isoforms was determined by RT‐PCR. Immunohistochemistry was performed to localize these isoforms in articular chondrocytes.

Key findings: All AC isoforms studied were found differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms increased with age.

Significance: These data add to the information about signaling cascades possibly involved in articular chondrocytes. Presence of AC isoforms 2, 4, 7, and 9 suggest a role for the cAMP mediated 2nd messenger signaling cascades in articular chondrocytes. Further studies, such as measuring the AC and cAMP levels during development and differentiation of articular chondrocytes would confirm our results and help in understanding the signaling mechanisms in articular chondrocytes.

Grant Funding Source: Supported by Seed money grant from the FHS‐Research Committee of the Aga Khan University, Karachi
3D imaging of the muscle fibers of the supraspinatus for improved rotator cuff repair (923.9)
Authors: Jorgensen Adam , Wisco Jonathan
Published: June 2021

The objective of this study was to construct a detailed 3D model of the muscle fibers of the supraspinatus to improve rotator cuff repair. To do so we have acquired n=2 embalmed cadaveric shoulders free of pathology. The shoulders have been imaged via MRI using T2‐Turbo Spin Echo (TSE) protocols with voxel sizes of 0.6x0.5x0.9 mm and 0.4x0.3x3.0 mm. The shoulders have been dissected exposing the supraspinatus. With the use of Microscribe 3D Digitizer we captured the intricate detail of the muscle fibers layer by layer throughout the mass of the supraspinatus. Using the image reconstruction program, Analyze, we spatially registered the MRI and Microscribe data to provide a contextually accurate, 3D depiction of the muscle fibers. More accurate depiction of the fibers is beneficial for understanding muscle fiber alignment during rotator cuff repair as well as postoperative healing.
A bologist's resource for plasmids: the DNASU and Protein Structure Initiative:Biology‐Materials repositories (928.1)
Authors: Seiler Catherine , Park Jin , Fiacco Michael , Throop Andrea , Steel Jason , Hunter Preston , LaBaer Joshua
Published: June 2021

The Protein Structure Initiative:Biology‐Materials Repository (MR; http://psimr.asu.edu) sequence‐verifies, annotates, stores, and distributes plasmids created by the Protein Structure Initiative (PSI). The DNASU plasmid repository (http://dnasu.org), which houses the MR, distributes over 198,000 plasmids created by our laboratory, by various consortia and by individual laboratories for distributions. This collection includes expression vectors, plasmid containing over 14,000 human genes, and as of November 2013, over 81,000 protein expression plasmids from PSI:Biology Centers. Through our recently redesigned website, researchers can search or browse our collection using our five search options or six new browse features which include an interactive vector browse and a collection browse that displays plasmids annotated by SMART domains. Detailed information about each plasmid such as the full length sequence, vector information, a dynamic vector map, and associated publications can be found directly on the website. Each plasmid also links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org) facilitating cross‐referencing of plasmids to additional protein annotations and experimental data. All plasmids can be requested through DNASU, and our facility ships to researchers worldwide. With the added biological focus in PSI:Biology, the distribution of these materials will help researchers expand the knowledge of the role of proteins both in normal biological processes and in disease.

Grant Funding Source: Supported by the National Institute of General Medical Sciences.
Identification of a DNA aptamer for the detection of sucralose in environmental water samples (928.4)
Authors: Robbins‐Welty Gregg , Parente Amy
Published: June 2021

Sucralose and many other non‐metabolizable food additives, drugs, and consumer personal care products are accumulating to detectable levels in our environment. Traditional analytical instrumentation or antibody‐based ELISA assays can quantitatively assess their presence and have been used to report detectable levels of sucralose in Lake Erie. Aptamers are DNA or RNA based molecules that adopt a specific structure that allows for a characteristic function, often the binding of a small molecule. Biosensors based on aptamer sequences thus provide a unique platform for detection of small molecules, generating a response to analyte through a binding‐specific conformational change. While aptamers have been developed for many target ligands, none exist for sucralose. SELEX (systematic evolution of ligands by exponential enrichment) is a methodology useful for selecting “winning” sequences ‐ the ability to adopt a shape that binds a target molecule ‐ from a library of possible sequences. The process involves several steps that ultimately separate and enrich the population of DNAs with sequences having a high affinity for sucralose. FLU‐MAG SELEX uses two specific modifications to traditional SELEX. Magnetic bead technology (“MAG”) will facilitate separation of mixtures of solutions while fluorescently labeled primers (“FLU”) will allow for the quantitative monitoring of DNAs as SELEX proceeds. Winning sequences can then be modified into structure‐switching biosensors capable of selective and sensitive detection of sucralose from environmental samples.
Elucidating the role of de novo mutations of cullin‐3 in hypertension (930.1)
Authors: Ibeawuchi Stella‐Rita , Agbor Larry , Quelle Frederick , Sigmund Curt
Published: June 2021

Loss of Cullin3 (Cul3) ubiquitin ligase activity is linked to hypertension (HT) attributable to increased RhoA protein in vascular smooth muscle cells (VSMCs). Patients with de novo mutations that lead to genetic deletion of exon 9 in Cul3 (Cul3Δ9) have early onset HT, but the mechanistic basis for this effect is lacking. We hypothesize that the Cul3Δ9 mutation results in reduced RhoA ubiquitination and degradation, thus elevating activatable RhoA protein levels. In support of this hypothesis, we found that exogenous levels of RhoA were increased in HEK293T cells cotransfected with Cul3Δ9 and the adaptor protein, Bacurd1, compared to cells expressing full length Cul3 (Cul3WT). Both Cul3Δ9 and Cul3WT were neddylated and both bound to Bacurd1. However, Cul3Δ9 binding to E3 ubiquitin ligase, RBX1, was decreased compared to Cul3WT. Dimerization of Cul3 ubiquitin ligase complexes is required for efficient ubiquitination of some substrates, and we show that dimerization of Cul3Δ9 is significantly impaired compared to Cul3WT. Thus, we conclude that Cul3Δ9 retains some activities of Cul3WT, but defects in Cul3Δ9‐associated ubiquitin ligase activity may impair RhoA ubiquitination thus stabilizing RhoA protein. Future studies will investigate the in vivo consequence of Cul3Δ9 on blood pressure and vascular function in a transgenic mouse model expressing Cul3Δ9 specifically in the VSMCs.

Grant Funding Source: Supported by NIH
Adaptor‐guided proteolysis initiates on the ribosome (932.1)
Authors: Puri Neha , Karzai Wali
Published: June 2021

Adaptor proteins extend the substrate range and specificity of AAA+ proteases. The SspB adaptor protein enhances the efficiency of ClpXP mediated degradation of tmRNA‐tagged proteins. We have investigated the mechanism by which SspB captures tag‐bearing proteins. Our inquiries reveal that SspB associates with tmRNA‐rescued ribosomes in a trans‐translation dependent manner, in a process reliant on distinct recognition signals within the appended tmRNA degron. Specific alterations in the tmRNA peptide sequence or the peptide‐binding groove of SspB have adverse effects on the efficiency of SspB ribosome association and the subsequent degradation of marked proteins. Our studies have also unveiled the ability of SspB to capture additional nascent polypeptides that harbor genetically encoded yet degenerate forms of the tmRNA degron and modulate their intracellular levels through directed proteolysis. Based on these findings, we propose that SspB scans translating ribosomes to capture signal‐bearing proteins for guided proteolysis by the ClpXP protease.

Grant Funding Source: Supported by NIH
Subcellular distribution of carboxypeptidase O affected by nutrient availability (933.1)
Authors: Lyons Peter , Dockery Donnel
Published: June 2021

Carboxypeptidase O (CPO) is a member of the M14 family of proteolytic enzymes. Many members of this family are secreted from cells and are involved in degradation of extracellular peptides; other carboxypeptidases remain within the secretory pathway where they are involved in the maturation of bioactive peptides. In order to determine the function of CPO, the subcellular localization of CPO was determined by immunofluorescence analysis. CPO, stably expressed in Madin‐Darby canine kidney cells, was found in a punctate pattern partially associated with lipid droplets. When cells were serum‐starved the structures associated with CPO became more numerous and often clustered. Addition of oleic acid to the medium caused CPO to be redistributed in a diffuse pattern with some concentration on the nuclear envelope. Western blot analysis of CPO following oleate incubation indicated an increase in the amount of a CPO isoform with lower mobility as seen by SDS‐PAGE, suggestive of a post‐translational modification. These changes in CPO size and distribution, dependent on nutrient availability, suggest a possible role for CPO in autophagy‐related processes.
Characterization of StiP, a starvation‐induced cysteine protease from Acinetobacter baylyi (933.4)
Authors: Daugherty Margaret , Lang Kristine , Lostroh C. Phoebe , Memmel Cody , Bernazzani Sarina , Reichert Blake , Dornbusch Amber , Arguella Joshua , Stanley Sarah
Published: June 2021

We have investigated the gene ACIAD 1960 from Acinetobacter baylyi. A. baylyi is a soil bacterium that is of interest due to its ability to utilize a diverse set of nutrients as a source of carbon and nitrogen, and to its high level of competence with regards to natural transformation. Medically, it is becoming of interest as a potential source of hospital‐acquired infections. Previous work has shown that gene ACIAD1960 is expressed during long‐term stationary phase. We identified that the purified protein expressed by ACIAD 1960 encodes a cysteine protease activity. We named the cysteine protease “StiP” to reflect that it is a long‐term stationary phase induced protease. We present data from a deletion‐insertion mutation which demonstrates that lack of the protein causes alteration of normal cell morphology, smaller twitching zone size, and hypersensitivity to the oxyanion tellurite. The mutant may also have a small growth defect. We show that the presence of divalent metal ions differentially affect its activity. The presence of Ca2+, Mg2+ and Mn2+ all increase activity, with Mn2+ showing the greatest effect. The presence of Zn2+ significantly decreases activity. Additionally, we show that StiP activity decreases in the presence of reducing agents, suggesting that a disulfide bond maycontribute to its overall conformational stability and function.

Grant Funding Source: Supported by Colorado College Natural Sciences Executive Committee grants to M.A.D., K.M.L., and C.
Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase (936.1)
Authors: Beckwith Robyn , Estrin Eric , Worden Evan , Martin Andreas
Published: June 2021

The 26S proteasome is the major protease in eukaryotic cells responsible for selective protein degradation to mediate protein quality control and regulation, yet the detailed mechanisms by which the proteasomal heterohexameric AAA+ unfoldase drives ATP‐dependent protein degradation remain poorly understood. Delineating the roles of the six distinct ATPase subunits in substrate processing has been hindered by limitations in working with endogenous proteasomes due to misassembly or lethal degradation defects. We therefore developed a heterologous expression system to produce the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro to perform systematic mutational analyses of the individual ATPase subunits. Our studies demonstrate that the six ATPases have distinct functions in degradation, corresponding to their positions in the spiral staircases adopted by their large AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits a pronounced three‐fold symmetry. Only three ATPase subunits, arranged in alternate positions within the unfoldase ring, contain a conserved C‐terminal hydrophobic/aromatic/unspecified (HbYX) motif that is critical for both peptidase binding and gate‐opening, whereas the C‐terminal tails of the interjacent ATPase subunits are dispensable. Our study provides an initial glimpse into the potential importance of the spiral staircase configurations of proteasomal ATPase subunits in substrate processing and highlights how the 26S proteasome may deviate from simpler, homomeric AAA+ proteases.

Grant Funding Source: Supported by US National Institutes of Health grant R01‐GM094497‐01A1
Allosteric regulation and substrate specificity of the proteasome deubiquitinase Rpn11 (936.2)
Authors: Worden Evan , Martin Andreas
Published: June 2021

The 26S proteasome is a multi‐protein complex responsible for ubiquitin‐mediated protein degradation in eukaryotic cells. Rpn11, the only essential proteasome deubiquitinase, removes ubiquitin chains from protein substrates during degradation, however very little is known about the regulation and intrinsic ubiquitin cleavage specificity of the enzyme. We utilized a heterologous expression system for the proteasome lid subcomplex, mutational analyses and in‐vitro deubiquitination assays to study the activity of Rpn11 in different assembly intermediates of the proteasome. We found that Rpn11 shows no preference for K48 or K63 linked di‐ubiquitin, indicating that it is promiscuous in substrate cleavage specificity. Furthermore, our studies have shown that Rpn5 inhibits Rpn11 in the lid complex and that this inhibition is relieved by incorporation of the lid into the proteasome holoenzyme, or by truncation of the Rpn5 N‐terminus. These observations support a model where the N‐terminus of Rpn5 blocks the Rpn11 active site in the isolated lid, but swings away from the enzyme to make contacts with the base and core particle upon formation of the proteasome. This allosteric regulation by Rpn5 may prevent spurious dubiquitination by Rpn11 in the isolated lid and thus ensure that the enzyme only acts on substrates committed to proteasomal degradation.

Grant Funding Source: Supported by National Science Foundation Graduate Research Student Fellowship
Regulating mtDNA dynamics by TFAM phosphorylation and degradation (94.3)
Authors: Suzuki Carolyn , Li Min , Venkatesh Sundararajan , Jayapalraja Thilagapathi
Published: June 2021

Mitochondrial transcription factor A (TFAM or mtTFA) is a central regulator of mtDNA replication as well as mitochondrial transcription. TFAM is also a major component of mitochondrial nucleoids, which are protein complexes bound to mtDNA that orchestrate genome maintenance, expression and inheritance. Despite the key roles of TFAM in mtDNA metabolism, little is known about mechanisms for its post‐translational regulation. Using a combination of biochemistry, cell and structural biology as well as proteomics, we have demonstrated that cAMP‐dependent protein kinase (PKA) phosphorylates TFAM within its high mobility group box 1 (HMG1), which impairs DNA‐binding and leads to rapid degradation by the mitochondrial ATP‐dependent Lon protease. We propose that reversible phosphorylation of TFAM within HMG1 causes electrostatic repulsion of the DNA phosphate backbone, thus providing a mechanism for regulating cycles of mtDNA binding and release. In addition, we speculate that irreversible degradation of DNA‐free TFAM by Lon rapidly fine‐tunes the abundance and function of TFAM at the mitochondrial genome.
Transcriptional properties of mammalian Elongin A and its role in stress response (944.1)
Authors: Inoue Makoto , Kawauchi Junya , Fukuda Mizue , Uchida Yohei , Yasukawa Takashi , Conaway Ronald , Conaway Joan , Aso Teijiro , Kitajima Shigetaka
Published: June 2021

Transcriptional elongation factor Elongin A exhibits an activity to repress the temporary pause of RNA polymerase II (RNAPII) on the DNA template. The role of Elongin A in RNAPII transcription in mammalian cells has not, however, been clearly established. In this study, we investigate the function of Elongin A in RNAPII transcription. First, we examined immunoprecipitation and immunostaining. These showed that Elongin A binds to the phosphorylated form of RNAPII (IIO) at both Ser5 and Ser2 and partially colocalized with the RNAPIIO and newly transcribed RNA. Significantly, Western blotting and qRT‐PCR showed that Elongin A was required for the maximal induction of stress response gene ATF3 and p21. ChIP assay showed that Elongin A occupancy was increased on ATF3, p21, c‐Myc and c‐jun genes after doxorubicin treatment. Furthermore, we performed re‐introduction assay of various mutants of Elongin A. It indicated that expressions of ATF3 and p21 gene were dependent on elongation activity of Elongin A. By contrast, polyubiquitination of RNAPII was dependent on ubiqutination activity of Elongin A. Taken together, our data provide the evidence that mammalian Elongin A is involved in the elongation phase of transcription in vivo and plays a crucial role in response to stress stimuli. Furthermore, our data indicated that two activities of Elongin A are independent of one another in response to stress.
Molecular basis of conversion of transcription termination factor NusA into an antiterminator (947.2)
Authors: Mishra Saurabh , Mohan Shalini , Godavarthi Sapna , Sen Ranjan
Published: June 2021

Abstract The well conserved bacterial transcription elongation/termination factor NusA is an essential component of the N‐mediated transcription antitermination machinery of the lambdoid phages. N‐NusA interaction during the transcription elongation process is mandatory for N to function. Mode of this interaction holds the key to understand the mechanism of conversion of NusA into an antiterminator by N. We have shownthat upon binding to the EC, the interaction surface of NusA for N changes from the NusA‐AR1 region to its NTD domain. We proposed that the close proximity of this N‐interaction site of NusA to its RNAP‐binding surface (β‐flap) enables N to exert conformational changes to RNAP allosterically via NusA. We further hypothesized the following. In the presence of N, i) NusA bind to a new site in the transcription elongation complex (EC) and/or, ii) NusA changes the conformations of the flap‐domain.In vitro competition assays revealed that the association of NusA to the EC stabilized several fold in the presence of N, which is indicative of an altered NusA‐EC interaction pattern. Two mutants, G1045D and R1058L, in the flap domain were known to affect N and Q‐mediated antitermination, respectively. These mutants unlike RNA‐exit channel mutants (P251S, P254L, G336S, and R270S; likely to be part of the N‐CTD interaction sites), were not suppressed by a suppressor, L108F, present in the RNAP‐binding domain of N. They were not‐defective for N‐mediated antitermination at the Rho‐dependent terminators, where N‐NusA‐RNAP interactions are less important. Finally, these two mutants exhibited severe synthetic defects with a NusA mutant, V8A, partially impaired for N‐binding. These results indicate that the antitermination defects of these β‐flap mutants were due to incomplete transition of flap domain by NusA in the presence of N, and thereby supported the aforementioned hypothesis.
The Escherichia coli Tol‐Pal complex is essential for the positioning of proteins at the cell poles (948.3)
Authors: Rajendran Madhusudan , Santos Thiago , Weibel Douglas
Published: June 2021

Organisms regulate essential biological processes, including growth, division, motility, and virulence, by controlling the subcellular localization of proteins and other biomolecules. Even though bacterial cells lack a complex internal system of endomembrane, they use numerous mechanisms to establish, maintain and replicate their subcellular organization. Particularly, the bacterial poles are home to numerous cytoplasmic and membrane proteins. However, the molecular mechanisms regulating the non‐uniform localization of polar proteins are largely unknown. Here we show that the Tol−Pal complex_a conserved component that maintains cell wall integrity in Gram‐negative bacteria_plays a key role in the polar localization of chemoreceptors in Escherichia coli. Mutants lacking the Tol−Pal complex and expressing fluorescently labeled chemoreceptors showed characteristically mis‐localized (i.e., non‐polar) chemoreceptors dispersed along the length of the cells. Cell motility and chemotaxis assays showed that the mutants presented motility and chemosensory defects that were not observed in the wild‐type strain. My long term goal is to investigate the role of the Tol−Pal complex in a diverse group of bacteria, with a focus on bio‐medically relevant organisms.

Grant Funding Source: Supported by National Institute of Health (1DP2OD008735‐01)
Generation of a new monoclonal antibody against zebrafish Insm1a (951.2)
Authors: Hoerter Jacob , Forbes‐Osborne Marie , Morris Ann
Published: June 2021

Zebrafish have the ability to regenerate many tissues including those of the central nervous system (CNS). The retina, the photosensitive tissue in the back of the eye, is an outgrowth of the CNS and has been shown to be capable of of regeneration after damage. The transcription factor Insm1 is expressed in the developing pancreatic and nervous system, neurogenic regions of the adult brain, and tumors of neuroendocrine origin. Additionally, in a model of chronic rod photoreceptor cell (PRC) degeneration, the progenitor cells involved in regeneration of rod PRCs express insm1a. Knock down of the retinally expressed zebrafish co‐ortholog of Insm1, insm1a, during embryonic development, resulted in delayed maturation of cone PRCs, and a loss of rod PRC differentiation.

Additional experiments are required to determine the mechanism by which Insm1 affects rod PRC differentiation during development, as well as its role in regeneration. Because Insm1 is a transcriptional regulator, it is important to know what downstream genes are regulated by Insm1. For these experiments, an antibody specific for zebrafish Insm1a is necessary. However, no suitable antibody is currently available. Thus, we decided to produce a monoclonal antibody against the entire coding region of insm1a. After cloning the protein coding region of insm1a downstream of a 6‐histidine tag, we used an inducible bacterial expression system to produce tagged Insm1a in vitro. The protein was successfully purified by affinity chromatography. After sequencing, the protein will be used as the immunogen in a monoclonal antibody screen. A purified antibody that specifically recognizes Insm1a will allow us to perform chromatin immunoprecipitation experiments to identify which promoters are regulated by insm1a. This information will help us to place Insm1 within a known transcriptional regulatory network and will provide information about the mechanisms by which Insm1 transcriptional regulation influences retinal development.
Identification of E6AP/UBE3A as a ubiquitin‐protein ligase that catalyzes encephalomyocarditis virus 3C protease ubiquitylation (952.1)
Authors: Notarianni Tara , Carmody Marybeth , Kirwin Katherine , Lawson T.
Published: June 2021

The 3C proteases of encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) are rapidly degraded by the ubiquitin‐26S protease system, which limits the 3C protease concentration that develops in infected cells. We have discovered that at least one UbcH7‐dependent ubiquitylation pathway is important for EMCV replication success determination. To identify the UbcH7‐dependent E3 ubiquitin‐protein ligase(s) involved in tagging the 3C proteases for destruction, we developed a protein purification scheme that begins with mouse cell lysate and ends with a ubiquitin‐UbcH7 affinity column. Analysis by mass spectroscopy of the proteins eluted from this column revealed the presence of four E3s, one of which is the HECT E3 E6AP/UBE3. The ability of this E3 to recognize the EMCV 3C protease as a substrate was confirmed using reconstituted mixtures containing purified E6AP. The major product of these reactions was monoubiquitylated 3C protease rather than polyubiquitylated conjugates. Whether this accurately reflects E6AP action in vivo remains to be determined, but it is possible that E6AP must function with other E3s to catalyze 3C protease polyubiquitylation in infected cells. Either UbcH7 or UbcH5 support E6AP‐catalyzed EMCV 3C protease ubiquitylation, with a slight kinetic preference for UbcH7. While the HAV 3C protease is also ubiquitylated by a UbcH7‐dependent E3, our results show that this E3 is not E6AP.

Grant Funding Source: Supported by NIH Award 1R15AI099134‐01.
β‐Actin is regulated by endothelin 1 and is not a consistent endogenous control in aortic smooth muscle cells from leptin‐deficient mice (955.1)
Authors: Carmo Luciana , Andrade Maria Claudina , Liberman Marcel
Published: June 2021

β‐actin, a highly conserved gene/protein, which is usually not modulated by different stimuli, is routinely used to normalize target gene or protein expression in experimental biology, and so defined endogenous housekeeping gene. However, investigators showed that β‐actin expression could change during cellular growth and differentiation. We hypothesized that endothelin 1 (ET‐1), a cellular proliferative agonist, increase β‐actin expression in vascular smooth muscle cells (SMC). Primary aortic SMC passage 4‐6 isolated from obese leptin‐deficient (obob) and from C57BL/6 mice were incubated without (Cont) or with ET‐1 50nM (ET) for 0‐72h. Cell lysates were assessed for β‐actin expression by Western blotting. ObobET SMC increased β‐actin expression after 24h, 48h and 72h (1.68 ±0.06, 1.66 ± 0.05, 1.73 ± 0.06 respectively) normalized by GAPDH vs obobCont (1.01± 0.07), p<.05 n=6. However, C57ET did not change β‐actin expression (0.91 ±0.06, 1.04 ± 0.03, 0.99 ± 0.08) after 24h, 48h and 72h respectively vs. C57Cont (0.95 ± 0.04), p=NS n=6. We showed that ET‐1 modulates β‐actin expression only in obob SMC, but not in C57BL/6 SMC. In conclusion, β‐actin is not a consistent endogenous control in ET‐1‐stimulated SMC from obob mice.

Grant Funding Source: Supported by FAPESP 2010/07455‐5
Investigation of potential as a functional food material of wild herb extract through sugaring (959.16)
Authors: Lee Sung Mee , Kim Dae Jung , Kang Yun Hwan , Kim Tae Woo , Lee Jae Sung , Choe Myeon
Published: June 2021

Many wild herbs are edible and that grow in the mountains or field areas. Also, wild herbs have diverse biological effects, such as antiobesity, anti‐cancer, antioxidant activities and immune stimulation. Currently, many studies about natural products are aimed at enhancing the efficacy of medicinal foods on biological activity using a bioconversion technology, including the fermentation or extraction process. In this study, the quality characteristics and antioxidative activity of the aged wild herb extracts were investigated. The quality characteristics were confirmed by extractability and sugar content (brix) measurement, and the antioxidant activity of aged wild herb extracts were assessed by the contents of polyphenol and flavonoid compounds and various radical scavenging assays using DPPH (2,2‐diphenyl‐1‐picrylhydrazyl), reducing power, and ABTS (2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid). Finally, through measurement of α‐glucosidase inhibitory activity suggests the potential antidiabetic functional activity of the aged wild herb extracts. Therefore, our results suggest that further studies are required to confirm the functionality that can be useful in the development of products.
Impaired tafazzin‐mediated remodeling of cardiolipin does not enhance susceptibility to ischemia and reperfusion injury (960.1)
Authors: Szczepanek Karol , Allegood Jeremy , Aluri Hema , Hu Ying , Chen Qun , Lesnefsky Edward
Published: June 2021

The elderly heart sustains increased injury during ischemia (ISC)‐reperfusion (REP) with a shift in the composition of cardiolipin (CL) in the Fischer 344 (F344) rat model of aging. The content and composition of CL, a phospholipid in the inner mitochondrial membrane, is critical for preservation of oxidative phosphorylation (OXPHOS) and inner membrane integrity. Tafazzin (TAZ) is an enzyme responsible for remodeling of immature CL with mixed acyl groups into the mature tetralinoleyl form (C18:2)4‐CL. We asked whether the defective remodeling of CL contributes to the increased injury in the aged heart. The elderly F344 rat heart expresses a truncated form of TAZ mRNA due to alternative splicing. We hypothesize that potential defects in TAZ‐mediated secondary remodeling of damaged CL is a mechanism of enhanced ISC‐REP injury in the elderly heart. We used the TAZ knockdown (TAZKD) mouse to test the susceptibility of decreased CL remodeling to ISC‐REP injury. TAZKD is an inducible system, where the expression of TAZ‐specific shRNA is controlled by tetracycline operator and can be induced by doxycycline (DOX). Only 1 day of DOX intake decreased TAZ mRNA in the heart to 20% vs. DOX‐treated WT. Knockdown was stable during long term feeding. CL phenotype was assessed by (C18:2)4‐CL content and was reduced 40% vs. WT at 2 mo. of DOX. TAZKD 2 mo. showed increased production of reactive oxygen species and increased susceptibility to calcium‐induced permeability transition at baseline, phenotypes observed in the aged heart. However, OXPHOS was unchanged. Infarct size, measured in isolated buffer‐perfused Langendorff hearts with 25 min. ISC‐60 min REP, was not altered in TAZKD hearts vs. WT. These results provide the first evidence that impaired TAZ‐mediated CL remodeling does not enhance susceptibility to ISC‐REP injury. Thus, age‐related defects in TAZ mRNA with the potential to impair TAZ activity in the aged heart are not a mechanism of the age‐enhanced ISC‐REP injury.

WT +2 months DOX
TAZKD +2 months DOX

TAZ mRNA (RQ/actinB)
1.00 ± 0.03 (n=4)
0.21 ± 0.01* (n=4)

(C18:2)4‐CL [pmol/mg]
12648 ± 590 (n=4)
7288 ± 962* (n=4)

H2O2 production [pmol/mg/min]
178 ± 18 (n=6)
241 ± 17* (n=6)

Calcium retention capacity [nM/mg]
1016 ± 70 (n=6)
536 ± 46* (n=6)

Glutamate+malate (complex I) [nAO/mg/min]
420 ± 32 (n=6)
421 ± 59 (n=6)

Succinate (complex II) [nAO/mg/min]
633 ± 80 (n=6)
537 ± 87 (n=6)

TMPD/ascorbate (complex IV) [nAO/mg/min]
1765 ± 190 (n=6)
1482 ± 186 (n=6)

Infarct size [% of LV]
29 ± 3 (n=5)
32 ± 3 (n=5)

Mean ± SE, *p<0.05 vs. WT.

Grant Funding Source: Supported by Department of Veterans Affairs Medical Research Merit Review Award.