bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of nutrient intakes among older adults with regional and racial differences
Authors: Hsiao Pao Ying , Mitchell Diane C. , Hartman Terryl J. , Jensen Gordon L. , Coffman Donna L. , Still Christopher D. , Allman Richard M. , Sawyer Patricia
Published: June 2021

Nutrient comparisons were made among subsets of older adults participating in the Geisinger Rural Aging Study (GRAS) (n=253) and the University of Alabama at Birmingham (UAB) Study of Aging (n=333) (mean age: GRAS=78.5 years, ± 3.8; UAB=72.4, ±5.21). The GRAS cohort is mainly Caucasian and rural. In contrast, the UAB cohort is 40% African American and includes both urban and rural participants. Preliminary data analysis, using t‐tests, revealed significant differences between blacks and whites in the UAB cohort (p<0.002) for intakes of key nutrients including energy, total fat, fiber, vitamin D, B12, calcium (Ca), magnesium (Mg), zinc (Zn), and iron (Fe). Consequently, successive analyses were completed comparing only the Caucasians in both cohorts. Significant differences (p<0.002) were found for almost all nutrients examined, including total and saturated fat, vitamin B6, B12, Mg, Fe, Zn, and Ca. Energy‐adjusted nutrient intakes tended to be higher in the GRAS cohort; however, both groups had inadequate intakes for key nutrients (fiber, vitamin D, B6, Mg, Fe, Zn, folate, and Ca). Subsequent analysis to compare dietary patterns will provide data to target specific foods to improve diet quality among diverse groups of older adults and will further our understanding of the association between food intake patterns, obesity and other health outcomes. USDA #1950‐51530‐010‐00

Grant Funding Source: USDA
The link between obesity, osteoporosis and cardiovascular disease
Authors: Chai Sheau C , Hooshmand Shirin , Saadat Raz L , Arjmandi Bahram H
Published: June 2021

Previously, we have reported that BMI can be used as a predictor of hyperlipidemia, hyperglycemia and inflammation in osteopenic postmenopausal women. Although evidence suggests a link between osteoporosis and cardiovascular disease (CVD), the extent to which obesity contributes to both conditions is unclear. The present study aims were to examine the association between bone biomarkers, serum ALP, CRP, BMI and CVD risk factors including lipid profiles and fasting blood glucose in this population. Overnight fasting blood and urine samples were collected from one hundred and thirty two (132) osteopenic postmenopausal women not on hormone replacement therapy. The Pearson correlation analyses were performed to examine the relationship among variables of interest. Our findings suggest that CRP is positively correlated with LDL‐C and triglycerides, while negatively correlated with HDL‐C. Serum total ALP was also positively correlated with BMI and a number of CVD risk factors including fasting glucose, Apo (B), TC, LDL‐C and Lp (a). Similarly, bone‐specific alkaline phosphatase (b‐ALP), a specific marker of bone turnover, followed the same pattern as did serum total ALP in reference to Apo (B), Lp (a) and BMI. These data suggest that serum ALP, b‐ALP, CRP and BMI can be used as predictors of CVD risk in postmenopausal women and perhaps these measurements should not be considered positive in bone studies.

Grant Funding Source: USDA
Impacts of dietary L‐arginine supplementation during early gestation (d 0–25) on the reproductive performance of gilts
Authors: Li Xilong , Bazer Fuller W. , Johnson Gregory A. , Burghardt Robert C. , Erikson David W. , Frank James W. , Spencer Thomas E. , Shinzato Izuru , Wu Guoyao
Published: June 2021

Between days 0 and 24 of gestation, 10 gilts per treatment were individually fed twice daily 1 kg of a corn‐ and soybean meal‐based diet supplemented with 0.0%, 0.4%, or 0.8% L‐arginine (Arg). The diets were made isonitrogenous by addition of appropriate amounts of L‐alanine (Ala). At day 25 of gestation, 30 min after consumption of Ala or Arg, gilts were hysterectomized to obtain conceptuses. Supplementation with 0.4% or 0.8% Arg increased (P<0.05) concentrations of Arg in maternal plasma (64% and 98%, respectively) as well as the vascularity of chorionic and allantoic membranes, compared with the control group. Numbers of corpora lutea (CL) and fetuses, placental and fetal weights, and embryonic mortality did not differ (P>0.05) between the 0.4% Arg and control groups. However, supplementation with 0.8% Arg decreased (P<0.05) uterine weight (−20%), total numbers of fetuses (−24%) and CL (−17%), total fetal weight (−34%), total volumes of allantoic and amniotic fluids (−34 to 42%), concentrations of progesterone in maternal plasma (−33%) as well as total amounts of progesterone (−35%), estrone (−40%), and estrone sulfate (−37%) in allantoic fluid, compared to the control group. Thus, supplementation with 0.8% Arg between days 0 and 25 of gestation, while enhancing placental vascularity, adversely affects the reproductive performance of gilts. Supported by USDA grants and TAMU Faculty of Nutrition.

Grant Funding Source: USDA
Mechanical ventilation and sepsis induce skeletal muscle catabolism in neonatal pigs
Authors: Orellana Renán A , Srivastava Neeraj , Gazzaneo María C. , Torrazza Roberto Murgas , Suryawan Agus , El‐Kadi Samer W. , Fiorotto Marta L. , Davis Teresa A. , Nguyen Hanh V.
Published: June 2021

Reduced rates of skeletal muscle accretion are a prominent feature of the metabolic response to sepsis in infants and children. Septic neonates often require medical support with mechanical ventilation (MV). The combined effects of MV and sepsis in muscle have not been examined in neonates, in whom there is normally rapid growth and protein deposition in skeletal muscle. Neonatal pigs (n=5–6/group) were subjected to MV and infused with endotoxin (LPS, 0 and 10 μg•kg−1•hr−1), dextrose, and a balanced amino acid mixture. After 9 hours, fractional protein synthesis rates (FSR) and translation and degradation signals were determined in the longissimus dorsi muscle. Compared to controls, MV alone decreased FSR by 22%, and by 36% in the presence of LPS. MV decreased eIF4G•4E association by 70% and by 95% in the MV+LPS group. The abundance of the E3 ligase, MURF‐1, was increased 6‐ and 9‐fold with MV and MV+ LPS respectively. These findings suggest that the MV‐associated induction of catabolism in peripheral skeletal muscle in neonatal pigs occurs by inhibition of protein synthesis and stimulation of protein degradation, appears aggravated by LPS‐induced sepsis, and promotes the skeletal muscle wasting associated with critical illness. NIH AR51563, NIH AR44474, and USDA 6250‐51000‐040

Grant Funding Source: NIH AR44474, and USDA 6250‐51000‐040
Effect of age and sex on whole body protein metabolism at dietary protein intakes that span the range of adequacy
Authors: Campbell Wayne W , Conley Travis B , Yarasheski Kevin E , Lim Eunjung
Published: June 2021

Purpose and methods

To assess the effects of age and sex on protein metabolism responses to dietary protein, 39 younger (Y)(n=21, 22–46 y) and older (O)(n=18, 63–81 y) men (M) and women (W) completed three 18‐d trials with protein (pro) intakes of 0.50, 0.75, or 1.00 g pro•kg BW−1•d−1. Fasting and fed state leucine (leu) kinetics were quantified on day 12 using a primed, constant infusion of 1‐[13C]leu.

Results

Independent of age and sex, higher pro intakes were associated with higher rates of fasting leu turnover (μmol leu•kg BW−1•min−1), oxidation and breakdown (P<0.05); synthesis rates were unchanged; and leu balance (balance; intake – oxidation) became less negative. Post‐feeding leu turnover, oxidation, synthesis, and leu balance increased and breakdown decreased. When expressed per kg fat‐free mass, fasting and fed state leu kinetics rates were not different between Y and O. In comparison to YM and OM, OW had higher fasting leu turnover, synthesis, breakdown, and balance, and higher leu turnover, synthesis, and balance after feeding.

Conclusion

Older women achieved higher fasting and fed state leu balances versus other groups when consuming protein intakes that span the range of adequacy. Reasons for age and sex differences and mechanisms explaining the more positive leu balance observed in the OW need to be explored. Support: NIH R01 AG15750, M01 RR14288, and RR000954, and USDA 98‐35200‐6151
Improved Overall Mood during Military Basic Combat Training (BCT), including lower Anxiety, Fatigue and Depression, is associated with changes in nutritional and metabolic state
Authors: Lieberman Harris R. , Kellogg Mark D. , Lesher Larry L. , Kramer F. Matthew
Published: June 2021

Relationships between mood and markers of nutritional and metabolic state, such as cholesterol, have been observed, but mechanisms accounting for these are obscure. During BCT, mood dramatically improves; anxiety, fatigue, confusion and depression decline over 60%. These changes occur rapidly and consistently due to the intense, structured nature of BCT, permitting an examination of relationships between concurrent changes in cognitive and metabolic state. We studied 35 females in U.S. Marine BCT, a physically and mentally challenging 12‐week course. Mood, assessed with the Profile of Mood States (POMS) questionnaire, and nutritional, metabolic and hormonal markers in plasma were assessed every 4 weeks. Multiple regression analyses demonstrated variation in HDL‐ and LDL‐cholesterol, fructosamine, triglycerides, free fatty acids, dehydroepiandrosterone‐sulfate, adrenocorticotropic hormone and substance P accounted for 44% of variation in anxiety, 40% confusion, 37% fatigue, 27% depression and 40% total mood occurring during BCT (p<.00001). Overall, positive changes in metabolic and hormonal status of female trainees during BCT accounted for a substantial proportion of individual variation in anxiety, fatigue and depression, as well as overall mood. We are not aware of previous work where a substantial proportion of human mood state could be accounted for by plasma markers. Supported by MRMC.
Early stimulation of vasopressin release maintains circulating blood volume in alcohol intoxicated rodents
Authors: Whitaker Annie M , Molina Patricia E
Published: June 2021

Acute alcohol intoxication (AAI) decreases mean arterial blood pressure (MABP), results in greater hypotension during hemorrhagic shock (HS), and impairs MABP recovery during fluid resuscitation from HS. The underlying mechanisms have been identified to include attenuation of neuroendocrine activation without alteration of pressor response. Whether AAI decreases blood volume, lowering tolerance to blood loss was not known. Chronically‐catheterized, adult male Sprague‐Dawley rats (250–300g) received a primed‐constant (2.5g/kg + 0.3g/kg/h) 15h intragastric infusion of alcohol or isocaloric/isovolumic dextrose (DEX). AAI resulted in an early and transient significant increase in urine output, decreased urine osmolality, and increased plasma osmolality. This was associated with a significant increase (43%; p<0.05) in circulating arginine vasopressin (AVP) 1h into the infusion. Blood volume and hematocrit were not different between the AAI and DEX groups at completion of the 15h infusion. These results suggest that AAI stimulates diuresis leading to increased plasma osmolality, which in turn stimulates AVP release decreasing urine output and restoring plasma osmolality. We conclude that decreased blood volume does not contribute to the impaired hemodynamic stability observed in AAI+HS. Supported by DOD PR‐054196 and NIAAA‐AA7577.
Hypoxic pulmonary vasodilation: a paradigm shift with a hydrogen sulfide mechanism
Authors: Olson Kenneth R , Whitfield Nathan L , Bearden Shawn E , St. Leger Judy , Nilson Erika , Gao Yan , Madden Jane A
Published: June 2021

Hypoxic pulmonary vasoconstriction (HVC), an assumed ubiquitous response of mammalian pulmonary arteries (PA), matches regional ventilation to perfusion via an unknown oxygen sensing mechanism. Global pulmonary hypoxia often produces pulmonary hypertension (PH) but PH is not observed in diving mammals where profound hypoxia is routine. Here we examined the response of cow and sea lion resistance (PA) to hypoxia and observed HVC in the former but a unique hypoxic vasodilation (HVD) in the latter. We then used this disparate response to examine the O2 sensing mechanism. In both animals, exogenous hydrogen sulfide (H2S) mimicked the vasoactive effects of hypoxia. H2S‐synthesizing enzymes, CBS, CSE and 3‐MST, were identified in lung tissue from both animals. The relationship between H2S production/consumption and O2 was examined in real‐time, using amperometric H2S and O2 sensors. H2S was produced by both sea lion and cow lung homogenate in the absence of O2 but it was rapidly consumed when O2 was present. Furthermore, consumption of exogenous H2S by cow lung homogenate, PA smooth muscle cells and heart mitochondria was O2‐dependent and exhibited maximal sensitivity at physiologically relevant PO2s. These studies show that HVC is not an intrinsic property of PA and provide further evidence for O2‐dependent H2S metabolism in O2 sensing. Support: NSF IOS 0641436 (KRO), NIH P20 RR016454 (SEB).
Effect of repeated water immersions on orthostatic tolerance and cardiovascular regulation during 70° head‐up tilt testing
Authors: Florian John P , Garner Christopher D
Published: June 2021

Water immersion (WI) reduces orthostatic tolerance (OT) and alters physiological responses to orthostatic stress, but the effect of repeated WI is unknown. Eight healthy men (35±2 yrs; mean±SEM) underwent 70° head‐up tilt (HUT) testing before and after 6‐hr O2‐breathing WI on 5 consecutive days (e.g. WI/1..WI/5). Heart rate (HR), blood pressure (BP: SBP, DBP, MAP), cardiac output (Q), limb blood flow (BF), stroke volume (SV), plasma volume (PV) and systemic vascular resistance (SVR) were measured during supine baseline (BL) and HUT. All data are compared to pre‐WI/1 BL values. Supine: HR and MAP were unchanged after repeated WI. Q, SV, and PV were reduced, and SVR increased after WI/1 (p<0.001). The drop in SV (~15 ml) persisted despite a return of PV to BL before WI/4 and/5. HUT: After WI/1, HR increased more than pre‐WI/1 (+32±3 vs +23±3 bpm) to compensate for lower SV, but SBP was reduced (−10±4 mmHg) (p<0.001). The tachycardic response to HUT was augmented over the week (+42±3 bpm, p<0.001), and SBP was further reduced after WI/5 (p=0.03), even though SV, SVR, and limb BF at post‐WI/5 were comparable to post‐WI/1. OT (max ΔHR during HUT) declined after WI/1 (p<0.001) and continued to diminish over repeated WI (p=0.02). We conclude that repeated WI exposures exacerbate immersion‐related reductions in OT. The responses suggest a shift in systemic and regional mechanisms to maintain BP and BF.

Support: ONR N0001409WX20220
Effect of exertional vs. passive heat stress on diurnal rhythms of hemodynamic parameters and parasympathetic modulation of cardiac function
Authors: Stauss Harald M , Liaboe Frederick O , Leon Lisa R , Kregel Kevin C
Published: June 2021

Previously, we demonstrated that the addition of exercise to exposure to high ambient temperatures (Ta), leading to increased core temperature (Tc), exaggerates alterations in autonomic nervous system function for several hours into recovery. Here we tested the hypothesis that this stress combination impairs diurnal fluctuations (DF) in hemodynamic and cardiac autonomic parameters. Rats were exposed to an Ta of 39°C while resting (HS‐C) or exercising on a running wheel (HS‐EX). Once Tc reached 41.5°C, the exercise protocol was stopped in HS‐EX and Ta was reduced to 25°C in both groups. Tc and systolic BP (SBP) were recorded telemetrically. Before the protocol, DF were apparent in Tc (+0.68±0.08°C), HR (+34±4 bpm), SBP (+6±4 mmHg), and relative high frequency spectral power of HR (HFHR, +6.4±1.5%). On the night and day following the experimental day, DF were preserved for Tc and HR in both groups. DF in SBP were preserved in HS‐C but not in HS‐EX. DF in HFHR were lost in both groups, suggesting that heat stress impairs cardiac autonomic function independent of the activity level. In conclusion, the lack of DF in SBP following heat exposure in HS‐EX but not in HS‐C suggests that the combination of heat stress and exercise causes greater cardiovascular impairment than heat stress alone, even with the same increase in Tc and shorter exposure to heat stress. (Supported by USAMRMC; author views not official US Army or DoD policy)
Altered substrate utilization during sequential short‐term under‐ and over‐feeding
Authors: DeGroot David W. , Ely Matthew R , Karl J. Philip , Young Andrew J.
Published: June 2021

Cycles of under‐ (UF) and overfeeding (OF) experienced during training and competition may result in alterations in energy expenditure (EE) and substrate utilization (SU) that impact body composition and physical performance. The time‐course and persistence of energy imbalance influences on EE and SU however, are not well‐defined. This study aimed to determine if a short period of UF affects resting EE (REE) and SU during rest and exercise during a subsequent period of OF. Volunteers (10M, 4F) underwent consecutive 4 day periods of energy balance (EB), UF (60% of EB) and OF (150% of EB); dietary macronutrient composition and total energy expenditure were constant. SU and EE were measured by indirect‐calorimetry on day 4 of each phase. REE was stable between conditions. During OF, resting fat oxidation was decreased (p<0.001) compared to EB and UF (0.32±0.29 vs. 0.93±0.51, 1.06±0.42, mg/kg‐FFM/min) and carbohydrate oxidation increased (3.30±1.02 vs. 2.00±1.43, 1.24±0.74 mg/kg‐FFM/min). Fat oxidation during exercise was higher during UF (2.39±1.29) and lower during OF (0.79±0.58) compared to EB (1.90±1.03 mg/kg‐FFM/min, p<0.001). Concomitantly, CHO oxidation during exercise was lower during UF (25.00±5.07) and higher during OF (29.69±5.98) compared to EB (27.11±5.27 mg/kg‐FFM/min, p<0.001). These data suggest that 4 days of energy imbalance is sufficient to alter SU during rest and exercise. Funded by MRMC.
Mitofusin 2 mRNA and mitochondrial DNA increase with exercise but not training
Authors: Dumke Charles L , Slivka Dustin R , Hailes Walter S , Cuddy John S , Ruby Brent C
Published: June 2021

Exercise increases mRNA for genes involved in mitochondrial biogenesis. A common measure of changes in mitochondrial capacity is the ratio between mitochondrial DNA (mtDNA) to genomic DNA (gDNA). In addition, little is known about the role of mitochondrial fusion in exercise training and mitochondrial development. The purpose was to examine the effects of acute and chronic exercise on mtDNA/gDNA ratio and mitofusin 2 (Mfn2) mRNA. Ten trained cyclists cycled ~100 miles a day for 21 days at a self selected workrate in the field. Muscle biopsies were obtained from the vastus lateralis immediately pre‐ and post‐exercise on days 1, 11 and 21. Muscle samples were analyzed for gene expression of Mfn2 and mtDNA/gDNA. Data were analyzed using a 2 (exercise) × 3 (day) repeated measures ANOVA. Exercise caused an increase in Mfn2 mRNA (P=0.038) and mtDNA/gDNA ratio (P=0.03). There was not a significant effect of day on either of these two outcomes (P=0.44 and P=0.99 respectively). Acute exercise caused an increase in the mRNA for Mfn2, a protein involved in mitochondrial fusion. This may explain the increase observed in mtDNA/gDNA following one 5 hr bout of exercise. Mitochondrial fusion‐fission may artificially alter the mtDNA/gDNA ratio making this measure difficult to interpret after exercise training.

Supported by AFRL, FA8650‐06‐1‐679 and the UM Grant Program.
Is glycosylation of aquaporins important for their membrane localization in gray treefrogs?
Authors: Goldstein David , Coffey Stephanie , Bella Paul , Frisbie James
Published: June 2021

In freeze‐tolerant Hyla chrysoscelis, cold acclimation and freezing involve accumulation of glycerol and distribution of glycerol and water among tissues and body fluids. Aquaporins, including HC‐1 (a water‐permeant protein) and HC‐3 (a glyceroporin conferring both water and glycerol permeability), facilitate those processes. These proteins are up‐regulated during cold acclimation, and glycosylated forms of the proteins are prominent. We hypothesized that glycosylation contributes to localization of the proteins in the plasma membrane. We tested this by fractionating liver tissue and examining the extent of glycosylation of aquaporins in membrane and cytosolic fractions, assessed by densitometry of Western blots with and without N‐glycosidase treatment of proteins. Specifics of glycosylation (banding patterns in Western blots) differed for membrane vs. cytoplasm, and for warm vs. cold. Preliminary studies suggest that, for HC‐1, expression in membrane was 3X that in cytoplasm in both warm and cold animals. Percent glycosylated HC‐1 was higher in membrane than cytoplasm, and in both fractions was reduced in cold acclimated animals. HC‐3 was about equally localized in membrane and cytoplasm in both warm and cold animals. We are as yet unable to detect an effect of acclimation on % glycosylation of HC‐3 (35–50% in both fractions, during both warm and cold acclimation). Supported by NSF IOB‐0517301.
The effects of feeding on the gas exchange pattern of Rhodnius prolixus
Authors: Contreras Heidy Lorena , Bradley Timothy J
Published: June 2021

Insects show discontinuous gas exchange patterns (DGC) when metabolic rates are low, however if metabolic rate increases, the insect gas exchange pattern changes to meet aerobic demand. We have previously demonstrated that an increase in metabolic rate, as a result of increased temperature, causes insects to transition from DGC to cyclic to a more continuous GEP. We report here on the effects of increasing metabolic rates through feeding. In the insect Rhodnius prolixus, metabolic rates can increase ten‐fold after a blood meal. These insects therefore provide an excellent model for studying the effects of feeding on insect GEP. We will report comparisons of GEP in Rhodnius at identical metabolic rates achieved by varying temperature or feeding, respectively. Our goal is to understand the relationship between metabolic rate and GEP.

Funding: APS Porter Physiology Development Award, NSF grant 105‐0920683
Inorganic phosphate (Pi) is actively transported from cerebrospinal fluid (CSF) by the choroid plexus (CP)
Authors: Renfro J. Larry , Guerreiro Pedro M. , Bataille Amy M.
Published: June 2021

The Pi concentration of CSF is about one‐half that of plasma in mammals, a phenomenon confirmed here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the role of CP in setting CSF [Pi]. The large sheet‐like IVth CP of the shark was mounted in Ussing chambers where unidirectional 33Pi fluxes revealed potent active transport from CSF to blood (Bl) side under short‐circuited conditions. The flux ratio was 25:1 with an average transepithelial resistance of 91 ± 18.9 Ω × cm2 and electrical potential difference of +0.9 ± 0.17 mV, CSF side positive. The active Pi absorption from CSF was inhibited by 10 mM arsenate, depolarization with 100 mM K+, 0.2 mM ouabain and by Na+‐free medium. Li+ stimulated transport 2‐fold compard to Na+‐free medium. Phosphonoformic acid (1 mM) had no effect on active Pi transport. RT‐PCR revealed both PiT1 and PiT2 gene expression, but no NaPiII expression, in the shark CP. PiT2 immunoreactivity was present by immunoblot, and immunohistochemistry localized the PiT2‐like transporter in the CP apical microvillar border. In conclusion, the CP actively removed Pi from the CSF. This process has transport properties consistent with a PiT‐type, Na+‐dependent transporter. A PiT‐like transporter is localized to the apical microvilli of the CP. Supported by NSF.
Bioengineered skeletal muscle for defect replacement in a rodent model
Authors: Machingal Masood Ahammed , Corona Benjamin T , Kesireddy Venu , Andersson Kristian , Herco Maja , Vishwjit Sonia , Bishwokarma Bimjhana , Zhao Weixin , Yoo James J , Christ George J
Published: June 2021

Loss of functional skeletal muscle due to congenital and acquired conditions, such as traumatic injury, tumor excision, etc., produces a physiological deficit for which there is still no effective clinical treatment. The goal of these studies is to develop clinically relevant tissue engineered skeletal muscle (TESKM) that can eventually be used for functional restoration of injured muscle in vivo. TESKM constructs developed by seeding rat muscle precursor cells on porcine bladder acellular matrix were preconditioned in a bioreactor for one week and implanted in nude mice (for 1–2 months) to repair a defect created by excising 50% of the native Latissimus Dorsi muscle (LD). Isometric tetanic specific force (ITSF) generated by the un‐repaired defect only group (49.2± 1.8 mN/mm2) was significantly lower (p<0.01) than that of native LD (161.1± 22.4 mN/mm2). ITSF of TESKM constructs increased over time from one month (20.06± 6.4 mN/mm2) to two month (86.75± 45.7 mN/mm2) which is 53% of native LD. Besides this, the TESKM constructs recorded higher tension than the unrepaired group (12.6± 4.1 g) both at 1 month (16.88± 7.1 g) and 2 month (22.34± 5.6 g, p<0.05), with the latter being 73% of native LD (30.67± 4.4g). Taken together, these initial observations indicate that TESKM can generate approximately half the ITSF of native LD after two months of implantation, illustrating the potential clinical utility of this approach.

Grant Funding Source: AFIRM
Saccharomyces cerevisiae responds to cold shock by inducing the transcription of ribosome biogenesis genes
Authors: Infanzon Bianca A , Buckmelter Kristen M , Liu Elizabeth M , Sakhon Olivia S , Citti Wesley T , Dahlquist Kam D
Published: June 2021

Previous studies on the global transcriptional response of budding yeast, Saccharomyces cerevisiae, to cold shock have revealed that the response can be divided into a set of early response genes (after 15 minutes to 2 hours of cold temperatures) and late response genes (after 12 to 60 hours of cold temperatures). The late response genes include the ESR genes induced by many environmental stresses, but less is known about the early response genes. We have extended these earlier findings to characterize more fully the early transcriptional response at 15, 30, and 60 minutes of cold shock at 13°C and also the response to recovery after cold shock for 30 and 60 minutes at 30°C using DNA microarrays. After 60 minutes of recovery from cold shock, gene expression returned to pre‐cold shock levels. Results were analyzed using the program GenMAPP to determine which biological pathways and processes were activated in response to cold shock and recovery. We found that genes involved in ribosome biogenesis were induced by cold shock and then repressed during the recovery phase. These results were confirmed through quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) experiments to detect the expression of known ribosome biogenesis genes such as NSR1. This work was supported by a Loyola Marymount University Kadner‐Pitts Research Grant (K.D.D.) and NSF award 0921038 (B.A.I., K.M.B., and K.D.D.).
A novel secondary alcohol dehydrogenase from Micrococcus luteus WIUJH20: purification, cloning, and properties
Authors: Huang Jenq‐Kuen , Park Jung Ki , Dhungana Babu Ram , Youngblut Nicholas D , Lin Chi‐Tsai , Wen Lisa
Published: June 2021

A novel NAD+‐dependent secondary alcohol dehydrogenase (2o‐ADH) has been purified to homogeneity from Micrococcus luteus WIUJH20 (M. luteus WIUJH20). It has a molecular weight of ~ 140 kDa as determined by gel filtration chromatography, and a molecular weight of ~ 34 kDa as determined by SDS‐PAGE. Thus, the enzyme is a homotetramer. The gene encodes for this enzyme has been amplified by PCR from the genome of M. luteus WIUJH20, cloned into pGEX2T expression vector, and recombinant protein overexpressed as a GST fusion protein (rGST‐2o‐ADH). Based on sequence homology, the enzyme is a NAD+‐dependent L‐3‐hydroxyacyl‐CoA dehydrogenase. But based on its catalytic activity, the enzyme belongs to secondary alcohol dehydrogenase. The rGST‐2o‐ADH fusion protein, the recombinant 2o‐ADH (r2o‐ADH) with GST‐tag removed, and the 2o‐ADH isolated from M. Luteus WIUJH20 had broad substrate specificity. Enzyme kinetics analysis showed that the r2o‐ADH possesses a Km and Vmax of 3.25 x 10−6 M and 150.3 μmol/min/mg respectively, when 12‐hydroxyoctadecanoic acid was the substrate. This r2o‐ADH prefers to use linear secondary alcohols as substrates over the cyclic secondary alcohols. The enzyme prefers long‐chain alcohol over short‐chain alcohol as shown by the decreasing in Km from 4‐heptanol (7 mM) to 6‐dodecanol (8 μM). Primary alcohols (particularly ethanol) are inhibitors to the 2o‐ADH and the inhibition constant Ki increases as the hydrocarbon chain length decreases.

The research was supported in part by grants from USDA from USDA CSREES 04‐35504‐14712 and 02‐015470, Western Illinois University Foundation, College of Arts and Sciences and the Honors College at Western Illinois University.
Enhancing the catalytic activity of an engineered threonine protease
Authors: Lansang Mie , Baird Teaster
Published: June 2021

In this study, the Cys‐42—Cys‐58 disulfide bridge of S195T trypsin was replaced with two valine residues to: 1) increase threonine protease activity and 2) better understand the structual limitations and requirements of this subsite with respect to catalysis. The activities of C42V/C58V and C42V/C58V/S195T trypsins (VV‐Tn and VVT‐Tn, respectively) were evaluated with benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) p‐nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) substrates. VVT‐Tn was 150 and 10 times more active towards the pNA and AMC substrates, respectively, than C42A/C58V/S195T trypsin (AVT‐Tn; kcat/KM values). In a thermostability study, both variants and WT trypsin retained > 80% of their activity suggesting that the substitutions did not destabilize the enzymes. The pH dependence of activity profiles were identical to WT trypsin, suggesting that any structural differences do not perturb the pKas of catalytically relevant residues. Finally, the KI of p‐aminobenzamidine was identical for native and VV‐Tn but that of VVT‐Tn was half the KI for WT. Therefore, Thr‐195 may affect the primary binding pocket structure. Collectively, these results indicate that the activity of Thr‐195 trypsin variants may be increased by appropriate substitutions at positions 42 and 58. However, the structural pertubations induced by these substitutions make achievement of wild‐type levels of activity less likely.

(This work was supported by NSF Award MCB‐0643988‐02)
Introducing extended substrate selectivity into trypsin through redesign
Authors: Najibi Sayeeda Phyllissity , Baird Teaster
Published: June 2021

The objective of this research was to evaluate how a residue with hydrogen bond capability may affect substrate selection by a previously engineered threonine protease. C42S/C58V/S195T trypsin (SVT‐Tn) was characterized and compared to trypsin variants C42A/C58V/S195T‐Tn (AVT‐Tn) and C42A/C58A/S195T (AAT‐Tn). p‐Nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) derivatives of benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) were use to kinetically evaluate the activity and specificity of SVT‐Tn. Their respective kcat/KM values show that SVT‐Tn is 8‐ and 40‐fold more efficient in Z‐GPR‐pNA and Z‐GPR‐AMC hydrolysis, respectively, than AVT‐Tn. Additionally, SVT‐Tn was 5‐fold less efficient in Z‐GPR‐pNA hydrolysis and 2‐fold more efficient in Z‐GPR‐AMC hydrolysis than AAT‐Tn. However, the (kcat/KM)pNA/(kcat/KM)AMC ratios show that SVT trypsin is 3.5‐fold more selective for the pNA substrate, less discriminatory than the AVT and AAT variants. The results suggest that a serine at position 42 in the S195T/C58V context significantly affects catalytic efficiency in a substrate dependent manner for the two substrates tested, but selectivity between the two substrates was only moderate. However, longer peptide substrates are needed to thoroughly evaluate the degree of the selectivity of SVT‐Tn.

(This work was supported by NSF Award MCB‐0643988‐02)
Single molecule structural dynamics of ribosome before and during translocation
Authors: Cooperman Barry S. , Chen Chunlai , Stevens Benjamin , Liu Hanqing , Cabral Diana , Zhang Haibo , Kaur Jaskiran , Wang Yuhong , Pan Dongli , Smilansky Zeev , Goldman Yale E.
Published: June 2021

We employ single‐molecule TIRF spectroscopy to study ribosomal structural dynamics over the first two elongation cycles, using complexes containing either Cy3‐labeled L11 and Cy5‐labeled tRNAs to monitor dynamics between L11 and P‐ or A‐site tRNAs (Lt complexes) or Cy3 and Cy5 labeled tRNAs to monitor dynamics between adjacent tRNAs (tt complexes). Each pretranslocation (PRE) complex has two FRET states, classical and hybrid, with similar transition rates between the two states. EF‐G.GTP converts PRE complexes to postranslocation complexes, giving a single new FRET peak in each case. Translocation occurs from both classical and hybrid states with similar probabilities, with no detection of a translocation intermediate (lifetime > 0.1 s) within our time resolution. EF‐G binding to the PRE complex strongly reduces fluctuations between classical and hybrid states prior to translocation. Moreover, a slow relaxation process seen in Lt complexes following the main translocation step is suppressed in the presence of the next cognate ternary complex, probably due to stabilizing interactions with the L11 region of the ribosome. Supported by GM080376 (NIH); 70NANB7H7011 (NIST)
GRK2 Activation By Receptors
Authors: Sterne‐Marr Rachel , Leahey Alex , Amie Sarah M , Donnelly Ryan , Lopez Thomas S , Tesmer John J
Published: June 2021

G protein‐coupled receptor (GPCR) kinases (GRKs) were identified by their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily and are activated by hormone‐treated receptors. Their mechanism of activation is largely unknown; thus, we initiated a study to identify GRK2 residues involved in interactions with their substrate receptors. Our study focused on the kinase large lobe and an extension of the kinase domain known as the C‐tail. Four substitutions, all within or adjacent to the C‐tail, resulted a decrease in the ability of GRK2 to phosphorylate activated GPCRs, rhodopsin and the beta‐adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of non‐receptor substrates. V477D had 12‐fold lower turnover number and was resistant to activation by agonist‐treated beta‐adrenergic receptor. Therefore, Val477 and other residues in the C‐tail are expected to play a role in the activation of GRK2 by GPCRs. Current studies focus on other regions of GRK2 required for receptor binding and activation. NSF Grants MCB0315888 and MCB0744739 to R.S.‐M. and NIH Grants HL086865 and HL071818 and American Heart Association Scientist Development Grant 0235273N to J.J.G.T.
Evidence for multiple active forms of the DNA damage response protein UmuD
Authors: Ollivierre Jaylene N , Wallace Alexandra , Beuning Penny J
Published: June 2021

All organisms experience DNA damage and have evolved multiple pathways to maintain genomic stability. In Escherichia coli, the SOS response to DNA damage involves the induction of 57 genes including the umuD gene products. The UmuD homodimer of 139‐amino acid subunits binds a RecA/ssDNA nucleoprotein filament which stimulates the latent ability of UmuD to cleave its N‐terminal 24‐amino acids, producing UmuD′2. UmuD2 and UmuD′2 play differential roles in the DNA damage response. Several models have been proposed for the conformations of UmuD2 including two versions with the N‐terminal arms in trans and two versions with the arms in cis (Beuning, et al. 2006 J. Biol. Chem 281:9633). These conformations expose different binding surfaces that may interact with other proteins. The goal of our research is to determine the conformation and dynamics of the umuD gene products in order to understand how their differential protein‐protein interactions regulate cellular responses to DNA damage. Our preliminary results suggest that a single amino acid substitution, N41G, weakens the KD for dimerization by over five orders of magnitude. This N41G mutation yields a UmuD monomer that is cleavable in the presence and absence of RecA/ssDNA. These results suggest that UmuD2 exists in multiple active conformations and that the cis conformation may be physiologically relevant. Supported by NSF Career Award MCB‐0845033.
DNA damage response protein UmuD displays conformational dynamics
Authors: Sikora Jacquelyn L , Ollivierre Jaylene N , Budil David E , Beuning Penny J
Published: June 2021

DNA damage is universal and all organisms have systems in place to respond to such damage. The Escherichia coli umuD gene products are essential to SOS‐induced mutagenesis in response to DNA damage. UmuD is a dynamic 139‐amino acid homodimeric protein that binds to a RecA/ssDNA nucleoprotein filament, which stimulates the latent ability of UmuD to cleave its N‐terminal 24‐amino acids to form UmuD’. The umuD gene products have been shown to perform distinct functions in both preventing and facilitating mutagenesis. Several models have been proposed for the conformation of the UmuD protein including two versions with the arms in trans conformation and two models with the arms in cis conformation. Each conformation exposes different binding surfaces through which UmuD could interact with other proteins. Through the use of models and structural data, it was concluded that structural rearrangement of UmuD occurs upon binding to the RecA filament and upon cleavage. The goal of our research is to determine the conformation and dynamics of the umuD gene products in order to understand how their protein‐protein interactions regulate cellular responses to DNA damage. Electron paramagnetic resonance spectroscopy (EPR) has been employed to probe the conformations of UmuD and UmuD’. Our EPR experiments suggest that the UmuD N‐terminal arms exhibit a large degree of motion. Research supported by NSF Career MCB‐0845033.
Comparing nematode enhancers: conservation, divergence, and co‐evolution
Authors: Ruvinsky Ilya
Published: June 2021

A major motivation behind the hundreds of genome projects is the notion that functionally important elements tend to evolve more slowly than other sequences, and can therefore be revealed by sequence comparisons. Comparative approaches have been highly successful in the past, but are we capturing the entire complexity of evolving genomes? We are using Caenorhabditis nematodes to address this question. We are combining sequence comparisons with direct experimental tests of functional conservation of orthologous enhancers. We find considerable disparity in the rates of sequence change in enhancers of co‐regulated genes and a poor correlation between sequence and functional divergence. While not completely negating the maxim “if conserved then functional and if functional then conserved”, our results suggest a greater role for relatively rapid and extensive co‐evolution within enhancers and between them and transcription factors. In turn, this suggests that a more nuanced approach will be required to extract information from comparative sequence data. This work was supported in part by grants from the NIH and NSF.
Markers of Bone Turnover, Osteoprotegerin (OPG), and Vitamin D across Gestation in Pregnant Adolescents
Authors: Essley Bridget V. , McNanley Thomas , Cooper Elizabeth , McIntyre Allison , Kent Tera , Foehr Marisa , Witter Frank , Harris Leah , O'Brien Kimberly
Published: June 2021

Pregnant adolescents may be at risk for vitamin D deficiency and bone loss across pregnancy. OPG is a soluble decoy receptor for RANKL, suppressing osteoclast differentiation and activity. To assess relationships between vitamin D, OPG and bone turnover, 25(OH)D, osteocalcin (OC; a marker of bone deposition), N‐telopeptide (NTX; a marker for bone resorption), and OPG were monitored in 88 pregnant adolescents (12–18 yrs) and their neonates. Blood samples were collected at mid‐gestation and at parturition when cord blood and placental tissue were also obtained. In these teens, OC (n=67), NTX (n=98), and OPG (n=66) were significantly elevated at delivery (p< 0.001). The gestational increase in OPG in these teens was larger than has been reported in mature women (p<0.001). Levels of NTX and OC were highly correlated (p<0.01) at mid‐gestation (n=87) and at delivery (n=75). At delivery, OC and NTX were significantly higher in neonatal than maternal circulation (p<0.001, n=69, n=84) while OPG was lower in cord versus maternal blood (p<0.001, n=67). Maternal vitamin D status was inversely related to OC at mid‐gestation (p<0.01, n=89) and at delivery (p=0.05, n=70). OPG was inversely related to vitamin D (p=0.01, n=69) in the neonate at birth. Recruitment and analyses are on‐going. Additional relationships between OPG, vitamin D status, and maternal bone loss and fetal bone accrual will be explored.

Grant Funding Source: USDA: 2005‐35200
Vitamin K Content of Hispanic Foods
Authors: Haytowitz David , Booth Sarah L. , Peterson James , Barakat Susan
Published: June 2021

To provide a food composition database for nutrition studies of Hispanics—the largest and fastest growing minority group in the United States—NDL initiated an increase in the number of Hispanic foods in the USDA National Nutrient Database for Standard Reference. In the first collections, samples of 21 foods were obtained from four Hispanic Community Health Study (HCHS) locations (Bronx, Miami, San Diego, and Chicago) plus the study diet center (Minneapolis). In 2009, a sampling plan with 12 locations, based on the Hispanic population of the U.S., was developed. Foods were identified from focus groups conducted with first generation immigrants from Cuba, Mexico, the Dominican Republic, and Puerto Rico, plus suggestions from the HCHS Diet Committee. Selected foods (19) are purchased from restaurants and markets in the 12 locations; composited at Virginia Tech and sent to the Vitamin K Laboratory at the USDA Human Nutrition Research Center on Aging for analysis. Phylloquinone content of the food samples was determined by reversed‐phase high‐performance liquid chromatography (HPLC). Phylloquinone values for plantain chips (27 mcg/100g) were higher than potato chips (22 mcg/100 g). Other foods, like empanadas, showed variation (2–13 mcg/100 g). These data will facilitate assessing the vitamin K status of Latinos in large national studies such as the HCHS and NHANES. Funding: USDA and NIH, Agreement No. Y1CN5010.
Dietary intake and plasma status of total carotenoids are inversely associated with depressive symptomatology in the Boston Puerto Rican Health Study
Authors: Ye Xingwang , Scott Tammy , Falcon Luis M , Tucker Katherine L
Published: June 2021

Antioxidant nutrients may play important roles in affective disorders, including depression. We examined associations of dietary intake and plasma status of total carotenoids with depressive symptomatology among 1216 Puerto Rican adults in the greater Boston area, aged 45 to 75 years. Depressive symptomatology was defined by Center for Epidemiologic Studies Depression Scale (CES‐D) score ≥16 or use of antidepressant medication. Intake of total carotenoids was inversely associated with CES‐D score (β = ‐4.2; P <0.001) after adjustment for age, sex, education, plasma cholesterol, vitamin B6, vitamin C and homocysteine, smoking, BMI and energy intake, using censored normal regression to account for antidepressant medication use. Similar association was observed for plasma carotenoids (β = ‐2.8; P <0.001). Further, both dietary intake and plasma status of total carotenoids were associated with lower risks of depressive symptomatology (odds ratios, 95% CI: 0.70, 0.51–0.95 unit of log‐transformed intake of total carotenoids [ug/d]; 0.82, 0.67–0.99 per μmol/L of plasma carotenoids) after adjustment for the same covariates. The inverse associations of intake and plasma status of total carotenoids with depressive symptomatology in this population suggest a protective effect of fruit and vegetable intake. Further population studies are needed to determine the causal direction. Supported by NIH P01 AG023394 and USDA ARS contract 58‐1950‐7‐707
Dietary cranberry, blueberry, and black raspberry affect the development of dyslipidemia and insulin insensitivity associated with metabolic syndrome in high fructose fed rats
Authors: Khanal Ramesh , Wilkes Sam , Rogers Theodore , Howard Luke , Prior Ronald
Published: June 2021

Effects of feeding cranberry, blueberry, and black raspberry powder on select parameters of metabolic syndrome were investigated in 40 growing male Sprague Dawley rats. The five dietary treatments were: 1) control AIN93G diet, 2) high fructose (65% by weight, HF) diet, and 3–5) HF diet with 5% (dry weight) of cranberry, blueberry, and black raspberry powder. Compared to control animals, HF reduced the body weight, abdominal fat, total liver lipids, and liver triglycerides. Urinary nitrate/nitrite excretion was not altered by diet (p>0.05). There was no difference among treatments in fasting and postprandial plasma glucose, uric acid or cholesterol concentrations, but plasma triglycerides were increased by high fructose feeding in both fed and fasted states. All three berries reduced fasting (p<0.01) but not postprandial plasma triglycerides. High fructose feeding increased fasting plasma insulin that was reduced by the berries. Values for homeostatic model of insulin resistance were increased (p<0.05) by HF diet but were reduced by the berries, while values for beta cell function showed a tendency to be reduced by the berries (p<0.09). HF feeding in a purified diet balanced for all nutrients did not produce a strong response in parameters associated with metabolic syndrome in growing rats, but all three berries were partially effective in ameliorating some of these parameters. Funded by USDA, ARS.
Total antioxidant capacity of grape and pomegranate juices in vitro is dependent upon assay dilution factor
Authors: Bolling Bradley Warren , Blumberg Jeffrey B , Chen C.‐Y. Oliver
Published: June 2021

Dilution is necessary to assess “Total Antioxidant Capacity” (TAC) within a linear range in vitro. However, the dilution factor (DF) has the potential to affect the accuracy of the TAC assay via altered interactions between its constituents. The effect of DF on the TAC of fruit juices has not been reported. We examined the effect of DF on TAC values of the ORAC, FRAP, DPPH, and Total Phenols (TP) assays in pomegranate juice (PJ) and grape juice (GJ). In the ORAC assay, a DF of 750 to 2000 enhanced GJ from 38 to 48 mM Trolox Equivalents (TE), respectively, but the same DF decreased PJ from 39 to 34 mM TE. Thus, at the lowest DF (750), the ORAC value of GJ was comparable to PJ, but at DF 2000, the ORAC value of GJ was 40% more than PJ. At equivalent ORAC values, PJ and GJ had similar kinetic patterns with a distinct lag phase, but had larger slopes of fluorescence decay than Trolox (−55 and −52 vs. −136 intensity/s). The TAC of a single DF at the midpoint of the standard curves for the respective assays were not significantly different from the average of 4 DF over the range of assay linearity. Increasing DF enhanced TAC values in the DPPH, TP, and FRAP assays but, in contrast to the ORAC assay, the degrees of change were not significantly different between juices. Therefore, the ORAC assay is more susceptible to the effect of DF than DPPH, FRAP, and TP assays. Supported by the USDA and POM Wonderful LLC. BWB was supported by NIGMS K12GM074869.