bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a role of p53, WWOX and TIAF1 as tumor suppression axis
Authors: Chou Pei-Yi , Lin Sing-Ru , Lee Ming-Hui , Hsu Li-Jin , Lin Yee- Hsin , Chang Nan-Shan
Published: June 2021

Tumor suppressor WWOX or WOX1 is frequently lost in many malignant tumors. Here we demonstrated that Wwox exon 1‐ deletion mouse embryonic fibroblasts (MEF Wwox−/−) migrated individually and faster than wild type Wwox+/+ cells. Normally, the wild type cells aligned together tightly and migrated collectively. WOX1 physically interacts with p53 and TIAF1 (TGF‐beta1‐induced antiapoptotic factor). Transiently overexpressed WOX1 suppressed the migration of breast MDAMB‐ 231 and MCF‐7 cells. Knockdown of WOX1 by small interfering RNA, or ectopic expression with dominant‐negative WOX1, increased the migration. While TIAF1 is an effector of the TGF‐beta/Smad signaling, TGF‐beta1 promoted Wwox+/+ MEF cell migration, but had had no significant effect on knockout MEF cell migration. Interestingly, TGF‐ beta2 increased wild type cell migration and suppressed knockout cell migration. Coexpression of WOX1, TIAF1, and/or p53 suppressed cancer cell migration and adherence‐independent growth. Together, WOX1 plays a regulatory role in TGF‐beta‐mediated cell migration. WOX1/p53/TIAF1 is a potential axis of tumor suppression. (Supported by DoD, USA, and NSC and NHRI, Taiwan)
Para‐aminobenzoic acid is an alternative aromatic ring precursor of coenzyme Q biosynthesis in mammalian cells
Authors: Xie Letian , Williams Kevin J , Marbois Beth , Tang Jeniffer , Bensinger Steven J
Published: June 2021

Coenzyme Q (ubiquinone or Q) is a lipid electron carrier in the electron transport chain. In yeast Saccharomyces cerevisiae nine genes, designated COQ1 through COQ9, have been identified as being required for Q biosynthesis. Generally, 4‐hydroxybenzoic acid (4‐HB) is considered the aromatic ring precursor to Q biosynthesis. Recently, para‐aminobenzoic acid (pABA), a well‐known precursor of folate, has been discovered as an alternative ring precursor to Q biosynthesis in yeast. This was a surprising finding because folate is synthesized de novo from pABA in yeast, but is a vitamin for human. In this study, we find pABA is also an aromatic ring precursor in Q biosynthesis in mammalian cells. We utilize liquid chromatography and mass spectrometry (LC‐MS/MS), and show that human and murine cells labeled with 13C6‐pABA synthesize 13C6‐Q. This work was supported by National Science Foundation Grant 0919609, National Institutes of Health (NIH) Grant GM45952, and Ruth L. Kirschstein National Research Service Award GM007185. The LC‐MS/MS determination of Q content was supported by Grant Number S10RR024605 from the National Center for Research Resources.
Comparing Diet Quality Based on a Dietary Screening Tool and the DASH Diet Index in Obese, Older Women
Authors: Beebe Nowen Elizabeth , Manganti Steven , Katkowski Lynn , Benson Marisa , Delmonico Matthew J , Lofgren Ingrid E
Published: June 2021

Diet quality impacts chronic disease risk and can be measured by the Dietary Screening Tool (DST) and the Dietary Approaches to Stop Hypertension (DASH) Index, however little research has compared these two methods. A 16‐week randomized clinical trial examined the additive impact of Tai Chi (TC) in obese, older women. Subjects completed a food frequency questionnaire (FFQ) and a DST. The FFQ output was converted to the DASH diet score. The DST provided a summary score. DST and DASH Diet scores were compared at baseline and post intervention using the following risk categories: <25th percentile as at risk, ≥25th
–<75th percentile as possible risk, and ≥75th percentile as not at risk. “At risk” and “possible risk” categories were combined and compared to “not at risk.” Thirteen women received modified DASH Diet Education (DE) and 14 women received TC in addition (TC+DE). Mean ages were 62.6 ± 5.9 y for DE and 60.4 ± 6.0 y for TC+DE. The number of participants in each risk category from the two questionnaires at baseline were compared and significant differences were seen as determined by fisher's exact test (p =.01). Though not significant, from baseline to post, those subjects classified as “not at risk” via DST increased from 4 to 15 versus from 5 to 7 via DASH score. Discrepancies between the classifications of risk may be because the DST measures diet patterns and the FFQ reflects nutrient intake.

Research supported by the USDA.

Grant Funding Source: USDA
Nutritional assessment of the School Lunch Program in Puerto Rico: Participants vs Non‐participants
Authors: Preston Alan M , Venegas Heidi , Rodriguez Cindy , Velez-Rodriguez Rose M
Published: June 2021

Extensive evaluations of the federally‐funded national school lunch program (NSLP) have been carried out on the U.S. mainland. Puerto Rico, a commonwealth of the U.S. but with an ethnically diverse population is an active participant in this program but has never been included in assessment studies. Multiple 24‐hr recall questionnaires were administered to groups of participating (P) and non‐participating (NP) children in the NSLP at 3 educational levels: elementary, middl and high‐schools. Comparisons were made for children within the study as well as between comparable children in mainland studies for total intake of several macro‐ and micro‐nutrients, contribution of the lunch to the total daily intake and adherence to U.S. Dietary Reference Intakes (DRI's) including acceptable macronutrient distribution ranges (AMDR's). Results show Both P and NP groups failed to reach RDA's for Vitamins A, D, E and calcium and exceeded levels for sodium, total fat and saturated fat Nevertheless, results suggest healthier intake of several nutrients in P vs NP which include increased Vitamin B12, folate, iron, zinc as well as less trans‐fatty acids. These findings parallel overall results from mainland reports. The unique feature of this study is that it is the first assessment of the NSLP in a completely Hispanic population. Supported by USDA, NRI Grant # 2003‐35200‐13590.
Sex differences in eating behavior during military training
Authors: Lutz Laura J , Karl J Philip , Cable Sonya J , Williams Kelly W , Young Andrew J , McClung James P
Published: June 2021

Initial military training provides an ad libitum feeding environment with controlled food choices. Sex differences in eating behavior during military training have not been investigated. Usual dietary intake was subjectively measured using a validated, quantitative food frequency questionnaire (FFQ). Male (n=83) and female (n=66) Soldiers (23 ± 5 yr [mean ± SD]) completed the FFQ at the beginning of basic combat training (BCT), reporting dietary intake over the previous 3 months, and at the end of BCT, reporting dietary intake over the 10‐wk course. In men, energy intake (EI) during BCT was greater than EI prior to the training (24.4 ± 13.4 v. 28.1 ± 10.7 kcal/kg, P < 0.05). The increase in EI was due to increased intake of carbohydrate from fruits, vegetables, and grains (P < 0.05). EI by women did not change during the training (28.6 ± 18.0 v. 27.2 ± 10.1 kcal/kg, P > 0.05). During BCT women increased fruit, vegetable and grain intake, but maintained total EI by decreasing fat consumption (P < 0.05). Calories from protein, and meat and dairy servings were unchanged during the training for both sexes; however females consumed less meat servings than males during the course (P < 0.05). Sex differences in eating behavior were apparent during BCT. These findings may suggest sex differences in eating attitudes and beliefs and the physiological response to military training. Research supported by MRMC.
Hand grip strength and estimates of body composition in HIVinfected rural Kenyan women
Authors: Ernst Judith Ann , Ettyang Grace , Johnson Cynthia , Siika Abraham , Nyandiko Winstone , Neumann Charlotte
Published: June 2021

Objective

To compare hand grip (HG) strength with estimates of body composition (BC) in HIV+ women.

Methods

In 256 drug naïve women (WHO Stage 1 or 2; CD4 cell count > 250 cells/uL), isometric strength was evaluated from HG measures using standardized technique and compared to BC estimates. The muscle areas of the mid upper‐arm, mid‐thigh, and mid‐calf, and sum of fat skin folds (SF) were calculated with standard equations using body circumferences and SF. Predicted values of fat‐free mass (FFM), % body fat and total body water (TBW) from bioelectrical impedance analysis were calculated with standardized equations.

Results

The mean (SD) for age and CD4 were 36.7(8.1) yrs and 533(229) cells/uL respectively. Significant correlations (p<0.001) were found between HG measures (right and left) and estimates of muscle area on the arm, thigh and calf, FFM and TBW and (p<0.005) between HG (left) and body mass index; correlations were not significant with fat mass estimates.

Conclusions

HG strength was related to estimates of lean mass at baseline, in drug naïve asymptomatic HIV+ rural Kenyan women enrolled in an 18 month randomized food intervention measuring the impact of beef vs soy vs wheat biscuit supplements on nutrition status. The trial is near completion, with positive acceptance of the biscuits. Supported by USAID Grant No. PCE‐G‐00‐98‐00036‐00, 1R01HD57646‐01A1 (CFDA #93.865) and Heifer Project International.
Maturation of protein kinase C masks its C1 domains
Authors: Antal Corina Elena , Skovsø Søs , Newton Alexandra C.
Published: June 2021

Protein kinase C (PKC) is important in proliferation and apoptosis, and its misregulation can give rise to cancer. PKC is processed by a series of ordered phosphorylations that are crucial in structuring it for catalysis; thus, understanding conformational changes that occur during maturation and activation of PKC are paramount. PKC contains tandem C1 domains that bind the lipid diacylglycerol (DAG), or the functional analogs, phorbol esters, an event that releases the autoinhibitory pseudosubstrate thereby activating PKC. Using FRET‐based methods, we show that unprocessed PKCs translocate to plasma membrane upon phorbol ester stimulation with a half time 6 times faster than wild‐type. This fast rate is mimicked by a construct of PKC's tandem C1 domains suggesting that both C1 domains are exposed in unprocessed PKC and become masked upon maturation. We propose a model for maturation in which unprimed PKC is in an open conformation with C1 domains exposed and the pseudosubstrate out of the active site. Upon maturation, PKC exists in a closed conformation with both C1 domains masked. Following intracellular Ca2+ release, PKC's C2 domain mediates translocation of PKC to membranes; subsequent binding of one of the C1 domains to DAG provides the energy to remove the pseudosubstrate, allowing phosphorylation of downstream targets.

This work was supported by NIH GM43154 to ACN, and NSF DGE1144086 and NIH T32 GM007752 to CEA.
Transient myogenic response provides insight to mechanotransductive pathways in vascular smooth muscle cells
Authors: Carlson Brian E. , Haydar Nada , Beard Daniel A.
Published: June 2021

The myogenic response, where vessels in the microvasculature can exhibit steady state constriction with an increase in intraluminal pressure, is facilitated through transduction of stress by vascular smooth muscle (VSM) cells. Traditionally the myogenic response has been quantified by recording steady‐state vessel diameters as a function of intraluminal pressure in isolated vessels. However this steady‐state response of vessel diameter provides less information than the complete transient response during these experiments. Our approach here utilizes transient response data of microvessels as they progress to steady state to uncover plausible mechanotransductive pathways. We have constructed a computational model considering vessel wall mechanics, VSM force generation and VSM Ca2+ handling and electrophysiology that is able to generate this transient myogenic response and we compare these simulations to transient response data from the literature. Through this comparison we can show that direct strain activation of ion channels (possibly non‐selective cation and/or L‐type Ca2+ channels) are likely responsible for the initial increase in cytosolic Ca2+ however slower mechanotransductive pathways (possibly mediated by cytochrome P‐450) must be present to explain the longer timescale transients in both cytosolic Ca2+ and vessel diameter observed experimentally. Supported by NSF CBET 1133260.
Neutrophil motion in straight and tapering P‐selectin‐coated in vitro models of lung capillaries
Authors: Tees David , Choi Young Eun
Published: June 2021

Neutrophil sequestration in lung capillaries is a key step in the inflammatory response to lung infection. Neutrophils were aspirated into capillary‐sized glass microvessels of varying diameters and taper rates coated with soluble P‐selectin or BSA. Cell velocity distributions were measured for pressure differences thought to be present across single pulmonary capillary segments. A creeping motion was observed with velocity significantly lower on P‐selectin than BSA, and that decreased with increasing P‐selectin concentration. The gap width between neutrophil surface and microvessel wall decreased with increasing concentration of P‐selectin while the force exerted by the bonds on the cell increased. It was also shown that high concentrations of anti‐P‐selectin F(ab΄)2 were effective to inhibit the P‐selectin mediated adhesion. These results demonstrate that even low densities of P‐selectin mediate neutrophil adhesion in the pulmonary capillary geometry. No mechanical arrest of neutrophils was observed in tapering vessels with taper angles of up to 9°, however, there is a transition point where cells slow down drastically, that does not depend on the P‐selectin concentrations, taper rate, or aspiration pressure. These results suggest that neutrophil arrest is primarily influenced by adhesion rather than mechanical trapping. This work was supported by grant BES‐0547165 from the NSF.
Hyperosmotic stress induces the myo‐inositol pathway by several orders of magnitude in tilapia (Oreochromis mossambicus) gill epithelium
Authors: Sacchi Romina , Gardell Alison M. , Kültz Dietmar
Published: June 2021

Myo‐inositol (MI) biosynthesis from glucose is one mechanism by which organisms can accumulate compatible organic osmolytes to counteract hyperosmotic stress. Here we show that the entire MI pathway is strongly induced by hyperosmotic stress in tilapia gill epithelium. We found that the second enzyme of this two‐step pathway, inositol monophosphatase (IMPA), is strongly up‐regulated by hyperosmotic stress at the protein level as shown by online LCMS. Using quantitative real‐time PCR we have investigated the level of induction of the MI biosynthesis pathway in gill epithelium after acclimating tilapia acutely and gradually from fresh water to either 34, 70 or 90 ppt salinity. Two distinct myo‐inositol‐phosphate synthase (MIPS) transcripts of the same gene showed similarly high levels of mRNA up‐regulation during all types of hyperosmotic stress. While only a single MIPS gene was found we detected at least two distinct IMPA genes in the tilapia genome. The IMPA1 gene that was highly up‐regulated after hyperosmotic stress at the protein level was also found highly up‐regulated at the mRNA level during all types of hyperosmotic stress. In contrast, IMPA2 was not up‐regulated suggesting that it lacks osmoresponsive enhancers. We conclude that the MI biosynthesis pathway represents a major hyperosmotic stress coping mechanism for euryhaline fish such as tilapia. Supported by NSF grant IOS‐1049780.
The energetics of oviparous reproduction in the African Brown House Snake
Authors: Jackson Alexander Garrett Schavran , Hicks James W.
Published: June 2021

The energetic cost of reproduction in the oviparous snake, Lamprophis fuliginosus (African Brown House Snake) was compared to other elevated metabolic states, specifically exhaustive activity and digestion. To determine gravidity, female snakes were imaged with a portable ultrasound (CTS‐3300, SIUI America, Inc.). Oxygen consumption was determined using flow through respirometry with carbon dioxide and oxygen sensors (Sable Systems International & AEI Technologies, Inc.). Results indicate that female L. fuliginosus exhibit a significant 29.6% (P=.0310) increase in standard metabolic rate while gravid. An additive effect of gravidity in female snakes during digestion and/or activity could not be measured. Compared to the metabolic cost of exhaustive activity (scope = 4.3; P<.00100) or digestion (scope = 6.70; P=.00100), gravidity (scope = 1.30; P=.0310) is significantly less energetically expensive. Furthermore, our data demonstrate that gravidity does not have a significant influence on time to exhaustion during exhaustive activity (mean = 28.6 minutes P=.400), or the time to peak digestive peak oxygen consumption (mean = 41.7 hours; P=.260). Digestion and activity are inevitable stresses a gravid female must endure. The potentially vulnerable time periods between ingestion and peak oxygen consumption during a meal, and the time it takes for a female to become exhausted during activity are not altered during gravidity. Besides a modest gravid metabolic scope of 1.30, our research has not identified any additional energetic physiological constraints. These results may have significant fitness implications for this species, which we plan to examine further in subsequent studies. Funding for this research was provided by NSF grant IOS 0922756 to JWH.
Removal of eggshell calcium impacts the development of corn snake embryos
Authors: Ross Whitney Trotter , Pyles Rebecca A , Stewart James R , Ecay Tom W
Published: June 2021

A fibrous and calcified shell characterizes eggs of oviparous amniotes and likely serves multiple roles during development. Calcium deposited on the outer shell surface is transported to the embryo late in development by the chorioallantoic membrane. Embryos of the egg‐laying corn snake Pantherophis guttatus acquire 28% of their calcium from the shell. To test the hypothesis that shell calcium is required for embryonic development, we reduced shell calcium by manually removing (peeling) the outer shell layer shortly after oviposition. Embryonic growth, calcium accumulation, bone ossification and chorioallantoic calcium transport protein expression were compared between treated and control groups. Hatchlings from peeled eggs were significantly shorter and lighter. Peeling delayed skull ossification (8 of 26 bones) early in development, although differences disappeared by hatching. However, hatchlings from peeled eggs showed significantly reduced ossification of caudal vertebrae. Calbindin‐D28K expression (marker for calcium transport activity) in the chorioallantois was reduced in peeled eggs. We conclude that reduced shell calcium retards growth of embryos and alters ossification patterns in corn snakes. Furthermore, the mechanism of calcium mobilization from the eggshell is responsive to variation in shell calcium content. (NSF grant IOB‐06515695. WTR supported by AOA fellowship.)
Physiological constraints on range expansion of the invasive, air‐breathing suckermouth armored catfish (Family: Loricariidae)
Authors: Fosha Kathryn Rose , Burggren Warren W
Published: June 2021

Members of the family Loricariidae, such as Hypostomus and Pterygoplichthys, are facultative air breathers and highly invasive in many North American freshwater and brackish systems. Despite management issues, little is known of their biological limiting factors and physiological ability to acclimate in new environments. Thus, environmental tolerances to temperature (10 – 38°C), salinity (0.5 – 37 ‰), and hypoxia (<21 – 2 kPa) were acutely evaluated. As temperatures were increased or decreased by 1°C day −1, starting at 25°C, fish fed normally between 18 and 38°C (9.4 – 11.5 g, n=6). Loss of equilibrium (LOE) occurred at a critical thermal minimum of 11.3 ± 0.6°C, but the higher temperatures were well tolerated. Both species (9.3 – 11.8 g, n=12) survived salinities up to 9.8 ‰ for 7 days (168 h), with little aerial breathing and no mortality. More frequent aerial breaths were seen in 18.7 ± 1.04 ‰, until LOE occurred after 130 ± 35 min. Fish introduced to seawater (35.2 ± 1.8 ‰) quickly lost equilibrium after 17.5 ± 7.7 min of exposure. Initial hypoxia tolerance tests on 1.5 – 2 g fish (n=6) determined that LOE occurred at approximately 2.6 ± 0.3 kPa in an average of 60.0 ± 10.7 min, without access to air. These wide tolerance ranges show that temperature, salinity and hypoxia levels found in southern North America do not strongly limit range expansion of invasive Loricariids. Research supported by NSF IOS‐1025823.
The effects of exercise and variable atmospheric oxygen concentrations on respiratory partitioning in the mudskipper, Periophthalmus modestus
Authors: Jew Corey J , Wegner Nicholas C. , Graham Jeffrey B.
Published: June 2021

Little is known about the physiological changes that occurred during the fish‐tetrapod land invasion (Middle to Late Devionian) because of the lack of extant members from this group. The mudskippers (Teleostei:Gobiidae) are an important proxy group for studying this significant evolutionary transition because, although distantly related, they too possess many aquatic respiratory and locomotive qualities that have been modified for use on land. Also, mudskippers vary in their degree of terrestrialilty and thus provide a comparison to different stages along the tetrapod progression. In this study, one species of mudskipper, Periophthalmus modestus, was used to assess the relative effectiveness of its two respiratory organs (buccopharyngeal chamber and skin) for aerial respiration during exercise recovery under varied oxygen concentrations (7%, 21%, and 35% O2) by measuring CO2 production (VCO2). Cutaneous respiration was found to correlate positively with O2 concentration and contributed the majority of total VCO2 under all conditions. However, the buccopharyngeal chamber contribution to total VCO2 was increased when skin rates were depressed (hypoxia) or when total rates were elevated following exercise. In hyperoxia, O2 diffusion rates across the skin were higher. This increased the cutaneous contribution and reduced the need for active ventilation, therefore lowering mudskipper respiratory costs. A hyperoxic atmosphere in the late Permian may have been similarly advantageous to early tetrapods. Hyperoxia could have increased the diffusion rate of O2 across the skin and the lungs making O2 more readily available, respiration more efficient, and increasing the aerobic capacity of the tetrapods as they adapted to life on land. This research was supported by a grant from the National Science Foundation.
Cold lungs, warm heart? Temperature effects on the oxygen‐hemoglobin dissociation curve of bar‐headed geese
Authors: Meir Jessica Ulrika , Milsom William K
Published: June 2021

Bar‐headed geese accomplish the extraordinary feat of migrating over the Himalayas, where oxygen (O2) levels are only ½ −1/3 that at sea‐level. It is well known that hemoglobin (Hb) of this species has enhanced O2 affinity, assisting O2 loading in this hypoxic environment. As temperature at these altitudes is extremely low and bar‐headed geese cross the mountains when air temperatures are at daily minima, we hypothesized that enhanced temperature effects may be beneficial for O2 loading (if these birds decrease temperature at the blood/gas barrier) and O2 unloading at the exercising muscle. Using the mixing technique on whole‐blood, preliminary evidence suggests that the O2‐Hb curve temperature coefficient of bar‐headed geese is higher than that of other birds near baseline CO2 (4%) and more interestingly, is highest when CO2 is high (7%) (e.g. at locomotory muscle). This should enhance unloading of O2 to exercising tissues, where temperature and CO2 levels will be near their maxima. At low CO2 (1%) (e.g. as may be experienced at the blood/gas barrier due to the enhanced hypoxic ventilatory response in this species) the temperature coefficient is lowest, suggesting that decreased temperature may not further enhance O2 loading. This finding parallels that of the diving emperor penguin in its analogous environment, as its air sac temperature is not decreased despite extremely low ambient temperatures. NSF Funded
Femoral growth plate is sensitive to loss of cardiac shunt in the American alligator
Authors: Owerkowicz Tomasz , Yang Johnny , Blank Jason M. , Eme John , Hicks James W.
Published: June 2021

Longitudinal growth rate (LGR) has been shown to correlate with calcified cartilage column height (CCC) in the growth plates of birds. In order to infer limb bone growth patterns in non‐avian dinosaurs, this correlation needs to be documented in crocodilians. We tested the LGR‐CCC correlation in femora of juvenile alligators raised for two years under laboratory conditions, and injected with fluorochrome dyes (alizarin, calcein). Some alligators were constrained to in‐series circulation by left aortic ablation (n=24), whereas sham‐operated controls retained in‐parallel circulation (n=36). Animals either remained sedentary or were subjected to rigorous walking or swimming exercise (n=20). We found CCC correlated with LGR and was significantly augmented (+36%) in alligators with in‐series circulation compared to those with in‐parallel circulation. Exhaustive exercise reduced thickness (−15%) of the distal, but not proximal, growth plate, suggesting local effect of biomechanic loading. We propose loss of the pulmonary bypass shunt results in higher systemic arterial oxygen tension, which allows for greater oxygen diffusion distance through the growth plate. Among archosaur ancestors, thicker growth plates may have appeared concurrently with the origin of in‐series circulation, and thus set the stage for later acquisition of fast growth and endothermic metabolism. Supported by NSF IOB 04445680 to JWH.
Effects of Hypoxia on Growth and Biomechanics in Limb Bones of Alligator mississippiensis
Authors: Lujan Susan L. , Owerkovicz Tomasz , Elsey Ruth M. , Hicks James W. , Middleton Kevin M.
Published: June 2021

Microanatomy and cross sectional geometry of limb bones are often used to infer life history and locomotor ability of fossil vertebrates based on study of their extant relatives; often the extreme variations in atmospheric oxygen that occurred over geologic time (oxygen levels 30% or more during the Permian, 15% in the Triassic) are not considered. As hypoxia is known to inhibit growth in vertebrates, while hyperoxia may be permissive, such comparisons between extinct and modern taxa merit investigation. As members of Archosauria, a clade with a long evolutionary history, alligators are excellent models to test for effects of environmental oxygen on skeletal growth and biomechanics. We incubated eggs and reared hatchlings in hypoxic (16% O2) and hyperoxic (26%, 31%, 36% O2) conditions. After sacrifice, femora were used in whole‐bone morphometric measurements and preparation of transverse mid‐diaphyseal ground sections. For comparison, traits were standardized to femur length. Cross sectional area, second moment of area, and polar moment of inertia scaled with isometry in alligators reared in chronic hypoxia, but with negative allometry in hyperoxic groups. The stouter femora of hypoxic animals exhibited greater resistance to compression, bending and torsion than predicted. Caution in evaluation of paleohistological data and consideration of oxygen availability is suggested.

Supported by NSF IOS‐0922756.
Identification of a nucleosomal region required for the proper distribution of the transcription elongation factor Spt16 across transcribed genes in Saccharomyces cerevisiae
Authors: Duina Andrea Angelo , Nguyen Trang Hoai , Wharton William , Harper Jennifer Aileen
Published: June 2021

In eukaryotic cells, DNA associates with several proteins to form a compacted structure referred to as chromatin. The basic unit of chromatin is the nucleosome, a particle consisting of a stretch of DNA wrapped around the histone octamer, itself composed of pairs of four core histone proteins: histones H2A, H2B, H3 and H4. In previous studies we described a mutant of histone H3 – H3‐L61W – that causes abnormal accumulation of Spt16 at the 3′ ends of transcribed genes. We now report that a mutation in histone H4 – H4‐R36A – causes a similar defect in Spt16 association with chromatin during transcription. Genetic evidence indicates that the H3‐L61W and H4‐R36A mutations affect Spt16 function through a common mechanism and an inspection of the crystal structure of the core nucleosome particle revealed that the H3‐L61 and H4‐R36 residues are located in close proximity to each other. These results suggest that a nucleosomal region defined by H3‐L61 and H4‐R36 plays a key role in ensuring proper Spt16 interactions with genes. We propose a model in which the region defined by these residues participates in a signal required for Spt16 to properly disengage from genes following the transcription process.

This material is based upon work supported by the National Science Foundation under grant No. 0919241.
Diaphanous 1 Negatively Regulates Cortisol Biosynthesis in H295R Human Adrenocortical Cells
Authors: Li Donghui , Wang Hongxia , Sewer Marion
Published: June 2021

Diaphanous 1 (DIAPH1) is a member of the diaphanous‐related forming family of proteins that modulate actin and tubulin polymerization. We have previously reported that DIAPH1 plays a key role in regulating mitochondrial movement and the production of cortisol and adrenal androgens in H295R human adrenocortical cells. DIAPH1 forms a macromolecular complex that coordinates inter‐organelle substrate delivery between mitochondria and the endoplasmic reticulum. In this study, we generated a cell line that stably expresses an shRNA targeted against DIAPH1. These cells exhibit increased mRNA and protein expression of multiple genes required for cortisol biosynthesis, including CYP11A1, CYP11B1/2, CYP17A1, and 3β‐hydroxysteroid dehydrogenase. Silencing DIAPH1 also induced the expression of steroidogenic acute regulatory protein, hormone‐sensitive lipase, and the melanocortin 2 receptor. Consistent with increased steroidogenic gene expression, DIAPH1 knockdown cells also secrete higher levels of cortisol than wild type H295R cells. Collectively, these results indicate that DIAPH1 negatively regulates cortisol production in H295R cells. This study is supported by NIH/NIDDK DK094151 and NSF MCB1005815.
Damage‐induced association of Rap1p with an internal binding site stimulates de novo telomere addition
Authors: Friedman Katherine Louise , Platts Margaret H. , Obodo Udochukwu C. , Paul Shira R. , Velkovsky Stoycho M. , Eli Ilham M.
Published: June 2021

At a low rate, sites of chromosome breakage in yeast are “healed” through telomere addition by the enzyme telomerase. While de novo telomere addition is normally repressed, it may serve to prevent chromosome loss in the face of irreparable damage. To address this possibility, we have sought factors that positively regulate telomerase action at chromosome breaks. Our study utilizes a “hotspot” for de novo telomere addition on yeast chromosome 5. This ~80 bp region contains TG‐rich sequences with similarity to yeast telomeric repeats. We find that function of the Rap1/Rif1/Rif2 complex is required for most de novo telomere addition within and flanking the hotspot. While this complex normally associates with telomeres to negatively regulate telomere length, Rap1/Rif1/Rif2 appears to stimulate telomere addition at ends with little or no telomeric sequence. Mutation of a putative Rap1p binding site at the centromere‐proximal end of the hotspot abolishes telomere addition, even though most of the TG‐rich sequences remain. Chromatin‐immunoprecipitation experiments demonstrate that Rap1p associates with hotspot DNA, but only after a chromosome break is induced telomere‐proximal to the hotspot. We suggest that Rap1p binds to internal chromosomal sites as a response to DNA damage and stimulates de novo telomere addition on a subset of broken ends to mediate damage tolerance.

Funded by the National Science Foundation.
Thermodynamic examination of U6 ISL bulged RNA
Authors: OˈConnell Allison , Grover Neena
Published: June 2021

A highly conserved bulge in the internal stem‐loop (ISL) of the U6 subunit of the spliceosome is responsible for its assembly and catalysis. This pH dependent bulge has an unpaired U80 nucleotide stacked within the helix that causes a bend in the RNA at pH 7.0. At a lower pH the flanking A•C base pair becomes protonated (forming an A+•C base pair) causing U80 to be flipped out into the major groove allowing the RNA to take up a conformation close to A‐form helix. We tested constructs containing the conserved bulged nucleotides as well as those with modifications in both the bulged and flanking helical nucleotides in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. Only constructs containing the A•C pair showed a gain in stability at pH 5.5 over 7.5. All constructs, with one exception, showed an increase in stability with increase in Mg2+ concentrations. RNA constructs also show greater gain in stability in the presence of Mg2+ than DNA constructs. Our results confirm that lowering the pH allows for the formation of an A+•C base pair in 1 M KCl and in 0.5 to 9.5 mM Mg2+ giving an extra stability of more than 1 kcal/mol in the wild type construct. This work was funded by NSF Grant MCB‐ 0950582 to NG.
Thermodynamic Examination of Small Symmetric and Asymmetric Loops Containing A•C and G•U Base Pairs in the Context of Group I Intron
Authors: Shishkova Evgenia , Grover Neena
Published: June 2021

Group I introns are large ribozymes capable of performing transesterification reactions to splice themselves out of an rRNA. Proper 3D assembly of the intron is crucial for its activation and formation of a correctly spliced product. Divalent metal ions, Mg2+ in particular, facilitate folding of the intron and are necessary for catalysis. Studies of RNA crystal structures report presence of metal ions in vicinity of highly conserved loops and bulges, suggesting that these regions play key role in functioning of the intron. We are examining the thermodynamic stability and metal ion binding properties of non‐canonical base pairs in the context of group I intron of Tetrahymena thermophile using small RNA as model constructs. An asymmetric adenosine‐rich tetranucleotide, a 4×1 A•C loop construct, and a symmetric 4×4 A•C loop construct were equally stable in 1 M KCl at various pHs. The 4×4 A•C loop gained more stability upon addition of Mg2+, as compared to 4×0 and 4×1 constructs. Protonation of A and consequent formation of A+•C base pair is expected at pH 5.5, and corresponding increase of 0.8 kcal/mol in stability of the 4×4 A•C construct was observed at pH 5.5 over pH 7. In 1M KCl at pH 5.5 and pH 7, a symmetric 4×4 G•U construct was 3 kcal/mol more stable than an asymmetric 4×2 G•U construct. The G•U containing RNA gained no additional stability in 9.5 mM Mg2+. This work was funded by NSF Grant MCB‐0950582 to NG.
Iron induced eukaryotic initiation factor/ mRNA binding affinity change
Authors: Ma Jia , Khan Mateen A. , Merrick William C. , Haldar Suranjana , Theil Elizabeth C. , Goss Dixie J.
Published: June 2021

Cellular iron metabolism is regulated by expression of iron responsive element (IRE) containing‐mRNA to control iron related protein levels (e.g. ferritin). The cytoplasmic iron regulatory proteins, IRPs, bind to iron response element (IRE) mRNAs, which represses IRE‐mRNA translation. IRE‐RNA binds to Fe2+ (in the absence of oxygen to prevent insoluble oxides) and the iron metabolite change the IRE‐RNA conformation resulting in decreased IRP (repressor) binding and increased eIF4F (enhancer) binding. eIF4F binds IRE‐RNA with high affinity (Kd=39.8±5.6nM) and further eIF4F: IRP competitively binds to IRE‐mRNA. eIF4G, a component of eIF4F, is also found to bind to the IRE‐RNA. The eIF4G middle domain binding affinity to the ferrtin IRE is lower than eIF4F and IRE binding doses not respond to metal concentration changes. While the eIF4G middle domain contains the RNA binding domain, additional protein domains and/or initiation factors (e.g. eIF4A, eIF4E) are necessary for functioning of the iron metabolite IRE‐ribo regulator.

This work is supported in whole or in part, by National Institutes of Health Grants DK20251 (D.J.G. and E.C.T.) and National Science Foundation Grant MCB 0814051 (D.J.G.)
Conditional protein splicing of inteins with a non‐canonical C‐terminal glutamine
Authors: Nicastri Michael C. , Xega Kristina , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Inteins are intervening polypeptides that self‐catalyze their own excision from the flanking polypeptides, or exteins, in a process called protein splicing. The reaction begins with an amide‐thioester rearrangement of the peptide bond that links the N‐extein to the intein. Next, the intein either proceeds to splice, or, if downstream steps are prevented, the thioester can be cleaved. We have examined inteins interrupting PolII proteins in both Methanoculleus marisnigri (Mma) and Methanospirillum hungatei (Mhu). Both inteins have C‐terminal Gln in place of the highly conserved Asn. We have observed that the inteins promote mostly N‐terminal cleavage with the native Gln, but promotes some protein splicing with mutation to the conserved Asn. Also, when expressed under oxidizing conditions, the Mma PolII intein accumulates unspliced precursor, which can be induced to undergo N‐terminal cleavage upon in vitro incubation with 1,4‐dithiothreitol. This suggests that a disulfide bond, perhaps between the terminal Cys residues, could regulate the initiation of protein splicing.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.
Non‐canonical inteins: Protein splicing by alternate mechanisms
Authors: Mills Kenneth V. , Reitter Julie N. , Connor Katherine R. , Nicastri Michael C. , Urbanski Laura M. , Williams Jennie E. , Colelli Kathryn M. , Savidis George , Marieni Michelle D.
Published: June 2021

Protein splicing is the post‐translational excision of an intervening polypeptide (intein) from its flanking domains (the exteins), concomitant with extein ligation. The first step of splicing is an amide‐to‐thioester rearrangement of the peptide bond linking the N‐extein and intein. Class three inteins bypass this step. The Clostridium thermocellum TerA intein is class three, whereas the Thermobifida fusca Tfu2914 intein is a traditional intein despite having class three sequence motifs. The third step of splicing is Asn cyclization coupled to cleavage of the peptide bond linking the intein and C‐extein. Both the Methanoculleus marisnigri (Mma) and the Pyrococcus abyssi (Pab) PolII inteins promote efficient splicing with C‐terminal Gln in place of Asn. The activity of the Mma PolII intein may be regulated by disulfide bond formation. The Pab PolII intein splices only at elevated temperatures, allowing for isolation of a stable precursor and study each step of splicing in vitro. An NMR solution structure of the Pab PolII intein reveals a particularly rigid structure, a disordered loop absent in a highly similar P. horikoshii intein, and a β‐hairpin specific to thermophilic inteins.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.
Characterization of a hypotaurocyamine kinase from the protozoan, Phytophthora sojae, and its implications on the evolution of substrate specificity in the phosphagen kinase family
Authors: Begres Brittany , Palmer Allyson , Van Houten Jason , Snider Mark , Fraga Dean
Published: June 2021

Phosphagen kinases (PK) are a family of enzymes that catalyze the reversible transfer of a high‐energy gamma phosphoryl group from ATP to a guanidino acceptor molecule. A novel hypotaurocyamine kinase (HTK) from the protozoan oomycete Phytophthora sojae (PsPK) was studied to determine substrate specificity, oligomerization state, and cooperativity. Isothermal titration calorimetry was used to determine the kinetic constants of PsPK reacting with taurocyamine and, less strongly, glycocyamine by titrating the guanidino substrate at varying concentrations into a solution containing the HTK and MgATP. The Km for taurocyamine and MgATP was found to be 0.59 mM and 3.78 mM, respectively, with a kcat of 34.66 s−1, while the Km for glycocyamine and MgATP was found to be 5.1 mM and 5.1 mM, respectively, with a kcat of 3.2 s−1. Size exclusion chromatography indicates PsPK is an oligomer and isothermal titration calorimetry suggests no cooperativity among subunits. Our results coupled with findings in Monosiga suggest oligomerization evolved first in the protozoa and that cooperativity sometime later. The data also indicate there is diverse substrate specificity in protozoan PKs previously thought to exist only in annelida. This project was made possible by the support of USDA grant (USDA 2011‐68004‐30104) and the Howard Hughes Medical Institute Undergraduate Science Education Program.
Redox Regulation of Rice Endosperm ADP‐glucose Pyrophosphorylase
Authors: Tuncel Aytug , Okita Thomas
Published: June 2021

The starch regulatory enzyme ADP‐glucose pyrophosphorylase (AGPase) is subject to allosteric regulation by the activator 3‐phosphoglyceric acid (3‐PGA) and inhibitor inorganic phosphate (Pi). The plastid‐localized enzyme is also subject to fine regulation by the redox state of the cells in response to light and sugar levels. The oxidized and low activity enzyme form contains an interchain disulfide bond formed by Cys12 residues between the pair of small subunits. Although these Cys residues are not conserved in the small subunit of seed (endosperm)‐specific AGPase, we provide direct evidence for redox control of cytosolic AGPase from developing rice endosperm. Biochemical analysis of the purified recombinant heterotetrameric AGPase showed that the enzyme is three times more responsive to 3‐PGA activation in the presence of reducing agent (A0.5 values, 0.8 vs. 2.9 mM). In addition, truncation of the large subunit by removal of N‐terminal peptide, a region which contains two conserved cysteines, resulted in loss of redox response of the enzyme (A0.5 values, 4.4 vs. 4.9 mM with and without DTT). Overall, our results show that the rice endosperm AGPase is controlled by a combination of allosteric regulation and redox control, the latter through modification of the large subunit instead of the small subunit as evident in the plastid‐localized enzyme. (This work was supported by DOE, grant number DE‐FG02‐08ER20216.)
Potassium channels on natural killer cells in the presence of breast carcinoma cells
Authors: Koshy Shyny , Wu Danli , Tajhya Rajeev , Hu Xueyou , Odejimi Abiola , Yotnda Patricia , Beeton Christine
Published: June 2021

Natural Killer (NK) cells are cytotoxic lymphocytes that play an important role in immune defense against viruses and cancer. Reduced NK cell activity has been associated with tumor resistance and metastasis in breast cancer. Our overall objective is to determine the phenotype and function of potassium channels on human NK cells in presence of breast cancer cells. We have found that human NK cells express two types of potassium channels, namely Kv1.3 and KCa3.1. The potassium channel phenotype did not vary with expression levels of CD16 and CD56 but it did differ between adherent and non‐adherent NK cells following cytokine‐induced activation. Blocking Kv1.3 and KCa3.1 channels differentially affected the proliferation, degranulation, and adhesion to fibronectin and tumor cells by adherent and non‐adherent human NK cells in vitro and differentially affected their ability to destroy tumors in vivo.

This work was funded in part by grants from the American Heart Association (0665009Y) and the Department of Defense (BC096018) to Christine Beeton.
Self‐polymerizing Zfra peptides elicit immune response for targeting cancer
Authors: Su Won-Pei , Lee Ming-Hui , Lin Sing-Ru , Chang Jean-Yun , Chang Nan- Shan
Published: June 2021

A novel immune response in targeting skin, breast, and many other cancer cells can be elicited by using self‐polymerizing peptides. Synthetic Zfra, a 31‐amino‐acid zinc finger‐like protein that regulates apoptosis, undergoes self‐polymerization in degassed enzyme‐free solutions. Pre‐injection of the full length Zfra1‐31, or truncated Zfra4‐10, via tail veins (<40 micromoles) protects nude mice from growing human lung cancer NCI‐H1299, basal cell carcinoma BCC, melanoma B16F10, glioma 13‐06‐MG and U87‐ MG, and many others. Alteration of Ser8 to Gly resulted in failure of self‐polymerization and cancer suppression, suggesting that Ser8 is central to the function of Zfra both in vivo and in vitro. When spleen cells from Zfra‐treated nude mice were transferred to naïve nude mice, these mice became resistant to the growth of implanted cancer cells (50–78% suppression), suggesting that non‐ T “memory” cells are involved in blocking cancer growth. NODSCID mice, which are defective in innate T and B cells, failed to generate anticancer response upon pretreatment with Zfra peptides. The observations suggest non‐T immune cells are involved in anticancer response. (supported by DoD, USA and NHRI and NSC, Taiwan)
The Role of PkcA in Septum Formation in Aspergillus nidulans
Authors: Henley Jordan Larissa , Jackson-Hayes Loretta , Hill Terry , Loprete Darlene , Dawodu Omolola , Delbove Clair , Poullard Ashely
Published: June 2021

Fungi of the Aspergillus genus can causes serious illnesses to those with compromised immune systems. PkcA in Aspergillus nidulans is important in formation of the cell wall and septa, which are both necessary for the invasive nature of the fungus. Elucidating PkcA's role in cell wall synthesis can be accomplished by understanding which domains facilitate specific functions of PkcA. Wild type PkcA localizes to growing hyphal tips and septation sites. To determine which domains are important for localization, green fluorescent protein tagged, domain deleted PkcA were constructed. The domains that are vital to A. nidulans maintenance of cell wall integrity were separately identified by growing the domain deleted stains in the presence of the cell wall stressor calcofluor white. We have determined that the localization signal and the domain responsible for proper function are distinct and essential domains.

This research was funded by NSF grant RUI‐0742907 and The Merck Institute for Science Education.

This research from the Jackson Heart Study is supported by NIH contracts N01‐HC‐95170, N01‐HC‐95171, and N01‐HC‐95172 provided by the National Heart, Lung, and Blood Institute and the National Center for Minority Health and Health Disparities
Isozyme‐specific interaction of protein kinase C δ with mitochondria dissected using live cell fluorescence imaging
Authors: Wu-Zhang Alyssa Xia , Newton Alexandra C.
Published: June 2021

Protein kinase C δ (PKCδ) signaling to mitochondria has been implicated in both mitochondrial apoptosis and metabolism. Using FRET‐based imaging, we observe that phorbol ester induces PKCδ to translocate to the outer membrane of mitochondria by an isozyme‐specific mechanism, resulting in increased mitochondrial respiration. A structure‐function study of the molecular mechanisms driving PKCδ's unique interactions with mitochondria reveals that the C1A and C1B domains and residues near the turn motif of PKCδ are required for its acute translocation to mitochondria and that the C2 domain, a critical charged residue in the activation loop, kinase activity, and mitochondrial transmembrane potential enable it to be retained there. PKCδ localized to the mitochondrial outer membrane has robust activity, as revealed by a FRET reporter of PKCδ‐specific activity (δCKAR). Perturbation of the kinase activity of PKCδ, by means of various kinase‐dead mutants, pharmacological inhibition, or a gate‐keeper mutant of PKCδ, demonstrates that its activity plays a role in both its translocation to and retention at mitochondria. These data support a model in which determinants unique to PKCδ drive a highly specific interaction with mitochondria. This work was supported by NIH GM43154 (ACN), UCSD Pharmacology Training Grant NIGMS T32 GM007752 (AXW), and UCSD GK‐ 12 STEM fellowship in education NSF 0742551 (AXW).