bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of 20 alpha‐HSD gene during bovine estrus cycle
Authors: Min Kwan‐Sik , Naidansuren P. , Park J.J. , Yun S.J. , Kang Y.S. , Kang Y.S.
Published: June 2021

The enzyme 20a‐hydroxysteroid dehydrogenase catalyzes the conversion of proesterone into its inactive form, 20a‐hydroxyprogesterone.Bovine ovary of corpus luteum were obtained at the slaughter. Placenta was also obtained at laparotomy under general anesthesia from a somatic cell nuclear transfer on day 280 of gestation. We determined the expression pattern of 20a‐hydroxysteroid dehydrogenase mRNA and protein by the real‐time polymerase chain reaction, northern, western blot, immunohistochemistry and immuno Flourescence. The mRNA and protein expression was highly detected in the corpus luteum 1 stage ovary. The molecular weight of rec‐protein produced in animal cells was founded in about 37 kDa by the expression vector pcDNA3aHSD. Thus, it may be could understand the function and role of bovine physiology during estrus cycle (ovulation mechanism), pregnancy and parturiton.

This grant was from BioGreen 21 program (#20080401034074), Rural Developmental Administration, Republic of Korea.
TACE‐induced cleavage of Neogenin disinhibits CNS axon/neurite growth by RGM in rat primary cortical neuron
Authors: Okamura Yusuke , Yamashita Toshihide
Published: June 2021

Neogenin is multifunctional receptor implicated in axon navigation, neuronal differentiation, morphogenesis, and cell death. Recently, neogenin appears to be a high‐affinity receptor for the repulsive guidance molecule (RGM), and RGMa‐neogenin interaction inhibits axon regeneration. While performing a two‐hybrid screen using the intracellular domain of neogenin, we observed that neogenin bound to ADAM17, also called TACE (TNF‐alpha converting enzyme). TACE is a 70‐kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases, and plays a role in proteolytic processing of some kinds of receptors, ligands, and enzymes. This process is of physiological importance. We show here that the cleavage of neogenin by TACE attenuates RGMa‐dependent neurite outgrowth inhibition in rat primary cortical neuron. Thus, TACE can be used as a molecular target for clinical conditions characterized by a failure of CNS regeneration
Paclitaxel‐induced growth inhibition in v‐Ha‐ras‐transformed NIH 3T3 fibroblasts is through the Spry2 down‐regulation linked to increased Raf‐1 kinase activity
Authors: Lee Michael , Ahn Jun‐Ho , Eum Ki‐Hwan
Published: June 2021

Paclitaxel is known to cause Raf‐1 kinase activation resulting in Bcl‐2 inactivation, which is a target substrate in pathways controlling apoptosis. We report here that v‐Ha‐ras‐transformed NIH 3T3 cells were significantly more sensitive to treatment of paclitaxel than its parental cells. This paclitaxel‐induced growth inhibition was found to be linked to increase Raf‐1/ERK activity. Moreover, Raf‐1 protein knock‐down makes cells more resistant to paclitaxel treatment. The expression of Spry2, which is known to modulate the Ras/Raf/MAPK signal either directly or through feedback regulation, was down‐regulated after the treatment of paclitaxel. As determined by analysis of Spry2 cytosine phosphate‐guanosine (CpG) island promoter methylation, down‐regulation of Spry2 appeared to be associated with altered methylation. Interestingly, siRNA‐mediated knockdown of Spry2 potentiated paclitaxel‐induced growth inhibition of v‐Ha‐ras‐Transformed NIH 3T3 cells. Taken together our results suggest that paclitaxel can induce growth inhibition by inducing high level of Raf‐1 kinase activity through the down‐regulation of Spry2.
Thrombin‐mediated thromboxane and 12‐HETE formation in human platelets is differentially regulated through PAR1 and PAR4
Authors: Holinstat Michael , Boutaud Olivier , Hudson W. James , Colowick Nancy , Young Summer , Oates John A. , Hamm Heidi E.
Published: June 2021

Thrombin‐regulated platelet activation mediates a diverse range of vascular effects which may result in thrombus formation and stroke. Thromboxane (TxB2) is thought to play a crucial role in the reinforcement of platelet activation following thrombin activation via TxB2 formation in the platelet. We demonstrate that PAR1 and PAR4 work synergistically but through unique signaling pathways following activation in order to generate both TxB2 as well as 12‐HETE. To investigate the signaling differences, we examined multiple biochemical and physiological steps involved in platelet activation including Rap1, aggregation, Ca2+ mobilization and formation of 12‐HETE and TxB2. Inhibition of PIP2 and PIP3 significantly altered PAR1‐, but not PAR4‐mediated platelet aggregation and resulted in inhibition of TxB2, but not 12‐HETE. Additionally, the time course for PAR1‐mediated TxB2 formation was faster compared to PAR4. Inhibiting PAR4 activation with YD3 only partially blocked thrombin‐mediated effects confirming the synergistic involvement of both PAR1 and PAR4 in this process. A better understanding of regulation of platelet function through PAR signaling pathways is crucial to developing a better class of anti‐platelet therapies with a lower profile for bleeding as a side effect. This study was supported in part by grants P50 HL081009 (to H.E.H.) and R00 HL089457 (to M.H.) from National Institutes of Health.
Rice isoflavone reductase‐like protein is associated with root development and ROS stress resistance
Authors: Kim Sang Gon , Kim Sun Tae , Wang Yiming , Kang Kyu Young , Lee Sang Yeol , Kim Sung‐Kun
Published: June 2021

Isoflavone phytoalexins and isoflavone reductase gene in the rice has not been identified and characterized. We characterized OsIRL protein using Northern, Western, 2‐DE, immunohistochemical analysis, and transgenic plant in rice. The transcript and proteins were more abundant in stems and roots than in flower and leaves. Expression of OsIRL was more accumulated in the root than in the shoot and seed. To investigate the release of reactive oxygen intermediates (ROI) by seed during germination, we utilized the apoplastic oxidation of 2¡‐,7¡‐dichlorofluorescin to fluorescent 2¡‐7¡‐dichloroflurescein and DAB staining as an in vivo assay. Expression of OsIRL was increased in the root elongation and maturation position carrying accumulation of ROI. Expression of OsIRL was increased in the oxidants (H2O2, FeCl3, MV) and decreased in the antioxidants such as reduced glutathione in the root and rice suspension cell. OsIRL was accumulated in the root sclerenchyma, outside of cortex, endodermis, and metaphloem cell. A certain concentration of H2O2 was necessary for root elongation, suggesting that OsIRL protein may play a role as antioxidant. The OsIRL transcripts levels were highly increased in callus, leaves, stem, and roots by treatment with ABA. The transgenic OsIRL plants have methyl viologen resistance, ROS‐generator, indicating that OsIRL have involved in root growth and resistance to oxidative stress.
Introduction to computer modeling using Jmol in an introductory biochemistry lab course
Authors: Johnson Warren Victor
Published: June 2021

It has been fifty years since the first three‐dimensional (3‐D) protein structure was published. There are tens of thousands of structures available in the protein databank. The manipulation of 3‐D structures can be very helpful to students seeking to understand protein structure and function. The easy to use, open source freeware, Jmol works well to enable beginning biochemistry students to examine structures of biomolecules.

Students in the first two sessions of a one‐semester biochemistry laboratory course quickly and easily learned to use Jmol to investigate the structures of various biomolecules in the protein databank. They prepared figures to illustrate particular features of an enzyme and prepared a report on the structure and function of an enzyme that included these figures. Later in this course students made use of this knowledge to design images for a poster presentation that explains the mechanism of an enzyme that they purified and characterized in the laboratory.

Students learned how 3‐D models are determined, how to access information from the protein databank, how to read and modify files in the database, how to view and manipulate 3‐D models with Jmol, and how to prepare figures in Jmol for insertion into a report. http://www.uwgb.edu/johnson
Free Amino Acid (FAA) Quantity in Human donor milk (DM) retained
Authors: Valentine Christina Jensen , Morrow Georgia , Fernandez Soledad , Gulati Parul , Bartholomew Dennis , Long Don , Welty Stephen , Rogers Lynette K , Nelin Leif
Published: June 2021

Background

Preterm infants are often fed donor milk (DM) when mother's milk is not available; however, there is a paucity of data on the free amino acid (FAA) profile of DM.

Objectives

Our hypotheses were that free amino acids would be effected by pasteurization and would have FAA levels different than in milk provided by preterm mothers.

Methods

38 donor milk samples were collected after informed consent and IRB approval (#0600532). Lactational stages 1‐10 months were combined into 16 pools to minimize variability. Free amino acids were measured pre and post PST by HPLC. Analysis was done using two sample t‐tests with unequal variances.

Results

Only two free amino acids were significantly different (p<0.0027) after PST of which Arginine was higher and Aspartate was lower. When comparing our PST samples to previous published amino acid means, our PST samples had significantly lower Glycine , Aspartate , Valine, Phenylalanine, Proline, Lysine, Arginine, Serine, whereas Histidine and Tyrosine were higher.

Conclusion

The finding that PST alters FAA concentrations minimally is reassuring but differences from previous fresh milk concentrations prompt further investigation into targeted lactational stage or amino acid supplementation for the donor milk fed immature neonate.
The association between hypertension and poor glycemic control with microalbuminuria among Cuban‐Americans with type 2 diabetes
Authors: Zarini Gustavo G. , Huffman Fatma G. , Perez Gomez Gianna , Nath Subrata D.
Published: June 2021

The study investigated the association between hypertension (HTN) and glycemic control (GC) with microalbuminuria (MAU) among 181 Cuban‐Americans with type 2 diabetes (T2D). Subjects were randomly recruited from Miami‐Dade and Broward Counties, FL. MAU was determined in urine samples by a semi‐quantitative assay. Blood pressure was measured twice and averaged. HbA1c was determined in fasting blood samples. Subjects were stratified into 2 groups according to HbA1c levels (<7.0% and >7.0%). Analyses used SPSS and included descriptive statistics and logistic regression controlling for age, gender, BMI, years with diabetes, smoking, kcal and protein intake, blood lipids, waist circumference, diabetes and cholesterol medications. Logistic regression analyses showed that after adjusting for the control variables, subjects with HTN were 2.44 times more likely to have MAU than those without HTN (OR=2.44, p= .036 95 CI 1.05 to 5.62). There were no significant differences among subjects with poor and good GC. Subjects with HTN and poor GC were 2.66 times more likely (OR= 2.66, p= .012, CI 1.24 to 5.70) to have MAU than those without HTN and good GC. The findings of this study indicate that the likelihood of having MAU is higher when hypertension and poor GC is present among Cuban‐Americans with T2D.
An Analysis of the Dietary Protein Intake of Hospitalized Elders
Authors: Amonette William Emil , Paddon‐Jones Douglas
Published: June 2021

Age related skeletal muscle loss results in reduction of strength and functional capabilities. During hospitalization physical activity is restricted, muscle loss is accelerated, and dietary protein intake becomes increasingly important as it represents one of the few means of upregulating muscle protein synthesis. The purpose of this study was to quantify the protein intake of hospitalized elderly patients in the ACE unit of a university hospital. Hospital meal plan data were entered into a nutritional software program and analyzed for nutrient content. The proportion of a single representative meal consumed was recorded for thirteen patients (77.5 ± 1.8 yrs). The hospital lunch meal provided 41.7 ± 2.0g of protein. However, patients consumed only 34.6% of the protein provided (14.4 ± 3.1g). Ten of the 13 subjects consumed less than 20g of protein (0.19 ± 0.04g/kg). The primary reason for low protein intake was an inadequate consumption of the animal protein menu item. In 6 of the 13 subjects, more than 50% of the protein ingested was attributed to the dessert item. Despite encouragement from dietary and medical staff, actual consumption was less than one‐third of the RDA for protein. If protein intake cannot be increased via provision of high quality protein‐rich foods, complementary nutritional strategies (e.g. amino acid supplementation) should be considered.
Healthy convenient prepared meals improve dietary quality during structured dietary intervention for heart disease risk factor reduction.
Authors: Nguyen Von , Summers Amber , Brosnahan Jennifer , Zukley Linda , Lowndes Joshua , Reimers Kristin , Angelopoulos Theodore J , Rippe James M
Published: June 2021

Caloric restriction is a typical approach for creating a negative energy balance for weight‐loss. Reduced food consumption may negatively affect dietary quality. Interventions that can maintain this in the face of caloric restriction are desirable.

We examined the effects of a DASH‐Style (DS; n=62) diet and exercise or exercise only (EO, n=49) program on cardiovascular risk factors for 24 weeks (BMI 22‐40). Both groups received an exercise prescription recommending a minimum of 15 minutes of moderate physical activity, 3 days per week and progressing to 45 minutes, 4 days per week by week 12. They also met with an exercise physiologist once weekly for 12 weeks and biweekly thereafter. Additionally, the DS group followed a reduce calorie eating pattern and incorporated one commercially prepared healthy frozen meal and one other selected grocery item each day for 24 weeks and received dietary counseling from a dietician once weekly for 12 weeks and biweekly thereafter.

After 24 weeks, DS reduced weight (‐18.04 ± 10.65 vs ‐6.05 ± 8.36lbs, p<0.001), intake of calories (‐486.0 ± 555.1 vs ‐42.7 ± 569.9kcal, p<0.001) total fat (‐39.0 ± 30.3 vs ‐5.2 ± 34.9g, p<0.001), saturated fat (‐16.1 ± 13.5 vs ‐3.0 ± 12.6g, p<0.001), cholesterol (‐123.6 ± 179.0 vs ‐38.1 ± 155.4g, p<0.01) and sodium (‐704.1 ± 1092.0 vs ‐41.8 ± 1224.6mg, p<0.01). Also, greater increases in fiber (9.8 ± 10.1 vs 2.8 ± 6.6g, p<0.001), soluble (3.2±3.2 vs 1.4 ± 2.5g, p<0.01) and insoluble fiber intakes (6.7±7.9 vs 1.1±5.5g, p<0.001) were observed in DS.

A structured calorie restricted diet including healthy prepared meals can help maintain, or improve, dietary quality thus reducing risk factors for chronic conditions.
Improved serum cholesterol concentration following a structured diet plan incorporating healthy convenient prepared meals
Authors: Brosnahan Jen , Summers Amber , Nguyen Von , Lowndes Joshua , Zukley Linda , Cooper Lisa , Reimers Kristin , Angelopoulos Theodore J , Rippe James M
Published: June 2021

Hypercholesterolemia is known to be a significant risk factor of coronary artery disease and is often accompanied by other lipid abnormalities including high triglycerides and low HDL. Interventions designed to improve the overall lipid profile are desirable.

We examined the effects of a DASH‐Style (DS; n=71) diet and exercise or exercise only (EO, n=60) program on cardiovascular risk factors in 131 participants (BMI 22‐40) for 24 weeks. Both groups received an exercise prescription recommending a minimum of 15 minutes of moderate physical activity, 3 days per week and progressing to 45 minutes, 4 days per week by week 12. They also met with an exercise physiologist once weekly for 12 weeks and biweekly thereafter. The DS group followed a reduced calorie eating pattern and incorporated one commercially prepared healthy frozen meal and one other selected grocery item each day for 24 weeks and received dietary counseling from a dietician once weekly for 12 weeks and biweekly thereafter.

After 24 weeks, the DS group reduced body weight to a significantly greater degree than EO (‐17.70 ± 10.11 vs ‐6.23 ± 8.12 lbs, p<0.001). In addition significantly greater improvements were observed in total cholesterol (‐12.01 ± 25.71 vs ‐1.75 ± 32.27 mg/dL, p<0.001). There were no other differences between the groups in lipid profiles.

These data suggest that a structured diet plan that incorporates healthy prepared meals can successfully improve measures of an atherogenic lipid profile and potentially reduce the risk for cardiovascular disease.
Regular leisure physical activity and food habits
Authors: Vicente Filipa , Pereira Paula Cardoso , Cabrita António Silvério
Published: June 2021

Regular physical activity is normally more easily associated with healthy food habits. The present study presents data of 14 assiduous gym members. The main goal is to evaluate if the physical active habits are combined with healthy food habits. The gym members had their weight and body fat mass measured in a Body Fat Scale (Tanita TBF‐300) and filled a questionnaire about food habits and a general food knowledge questionnaire.

The anthropometric evaluation showed that 57% of this small sample was within their recommended weight range and healthy body fat percentages. Even so, 2 individuals were obese. The vast majority (78%) doesn't drink alcoholic beverages except in special occasions and there was just one smoker in the group. They choose foods mainly because there are nutritional balanced (43%) or healthy (21%) and the food habits and frequency questionnaire revealed a low sugar, salt and processed meats consume. General food knowledge is poor in what concerns to different types of fat and recommended fruit and vegetable portions.

This group already endorses some healthy food habits; further evaluations of more gym members are needed in order to evaluate intervention needs in these individuals.
Diabetes Self‐Management Cuban Americans
Authors: Vaccaro Joan A , Huffman Fatma G , Zarini Gustavo , Nath Subrata
Published: June 2021

Objectives

We investigated the relationship among factors predicting inadequate glucose control among 182 Cuban‐Americans (Females=110, Males=72) (CA) with type 2 diabetes mellitus (T2DM).

Methods

Cross‐sectional study CA adults self‐reported with type 2 diabetes (T2D) from a randomized mailing list in three counties of South Florida. Participants were screened by trained interviewers for heritage and diabetes status and participants signed informed consent form. Statistical analyses used SPSS and included descriptive statistic, multiple logistic and ordinal logistic regression models.

Results

Only 54% reported having a diet prescribed/told to schedule meals. Those told to schedule meals were 3.62 more likely to schedule (CI 1.81, 7.26) p<0.001. Age, WC, BMI and gender were not significant predictors of HbA1c. We found, by ordinal logistic regression of 5‐scale Likert, that participants with inadequate glucose control (HbA1c> 8.5) were more than four‐fold likely to never follow a meal plan , OR 5.39 (1.40, 20.7).

Conclusions

Nearly half CA are not given dietary guidance, yet our results indicate CA may improve glycemic control by receiving dietary instructions.

Grant Funding Source

American Diabetes Association
Socioeconomic factors associated with obesity in adolescents in Guadalajara, Mexico
Authors: Romero‐Velarde Enrique , Martínez‐Gloria Esther Alicia , Vásquez‐Garibay Edgar Manuel , Troyo‐Sanromán Rogelio
Published: June 2021

Objective

To identify differences in socioeconomic factors associated with obesity in adolescents from different social classes.

Methods and materials

In a cross sectional study 328 adolescents from three junior high schools were included; 131 from low, 106 from middle and 91 from high social class. Socioeconomic information was obtained; the body mass index (kg /m2) was used to evaluate the presence of obesity (BMI >95 percentile) or healthy weight (BMI percentile 5‐85). The association between the presence of obesity and socioeconomic characteristics was measured with the odds ratio (OR) and x2 test.

Results

The prevalence of obesity was higher in middle (24.5%) than low (18.3%) or high class schools (19.8%). There was an association between the presence of obesity in adolescents and mother's obesity in schools of low (OR = 2.77) and middle (OR = 3.0) social classes and with father's obesity in schools of middle (OR = 2.81) and high (OR = 3.62) social classes. The practice of physical activities was a protection factor for obesity in adolescents from low social class (OR = 0.28 [0.10‐0.76]).

Conclusions

The prevalence of obesity was higher in adolescents of middle social class; probably, the highest prevalence is moving from higher to lower social classes in Mexico. Adolescents from low social class might have less oportunities to practice physical activities and look for health attention.
Hyperglycemia and hypertriglyceridemia were associated with altered hepatic energy regulation in rat offspring from fructose fed dams
Authors: Zou Mi , Teegarden Dorothy , Donkin Shawn S.
Published: June 2021

Maternal nutritional stress acts to program metabolic syndrome in offspring. Dietary fructose is linked to metabolic disorders. We hypothesized that maternal fructose intake would impact energy metabolism in offspring. Future, we hypothesized that these effects would be magnified by postweaning fructose intake. Sprague‐Dawley rats (n=8) were fed either a control (C) or a 63% fructose diet (F). At birth pups were cross‐fostered onto dams fed diet C. Pups within maternal diet group were fed either C or F diet after weaning to give maternal × postweaning diet combinations: CC, CF, FC, and FF. Pups showed greater (P<0.1) blood glucose for FC and FF compared to CC and CF (51.7, 49.0, 45.7, 45.8±2.1 mg/dl) at 114 days of age. Hepatic fatty acid synthase (1.52, 1.90, 0.02, 0.01±0.44), carnitine palmitoyltransferase‐1 (3.9, 5.2, 1.4, 1.0±0.8), pyruvate carboxylase (0.36, 0.48, 0.04, 0.05±0.07) and PPAR? coactivator‐1a (2.3, 3.3, 1.7, 1.2±0.6) mRNA levels were elevated for FC and magnified for FF pups at 130 days compared to CC and CF. Serum triglyceride reflected similar changes (32.0, 43.5, 17.0, 29.2±7.5 mg/dl, FC, FF, CC, CF). Maternal fructose intake leads to hyperglycemia, hypertriglyceridemia and elevated expression of genes for hepatic energy metabolism in offspring; changes that are associated with metabolic syndrome. These effects are further magnified by postweaning fructose intake. Supported by NIH DK077581.
Energy expenditure in chow‐fed female non‐human primates of various weights
Authors: Rising Russell
Published: June 2021

Objective

In this study daily energy expenditures (EE) and respiratory quotients (RQ) in female monkeys of various body weights were determined.

Methods

16 socially reared Bonnet Macaque female monkeys [5.5 ± 1.4 kg body weight, modified BMI (length measurement from head to base of the tail) = 28.8 ± 6.7 kg/crown‐rump length, m2 and 11.7 ± 4.6 years] were placed in the primate Enhanced Metabolic Testing Activity Chamber (Model 3000a, EMTAC Inc. Fort Lee, NJ) for 22‐hour measurements of EE (kcal/kg) and RQ (VCO2/VO2). All were fed monkey chow (4.03 kcal/g) ad‐libitum under a 12/12 hour light/dark cycle. Metabolic data were corrected for differences in body weight. Results were divided into day (8‐hours), dark (12 hours) and morning (2‐hours) periods. Data analysis was conducted utilizing SPSS (Version 13).

Results

Modified BMI negatively correlated with 22‐hour energy expenditure in all monkeys (r=‐0.64, p<0.01). The large variability of daily energy intake (4.5 to 102.0 kcal/kg) necessitated division into two groups, non‐eaters (<13 kcal/kg) and eaters (>23 kcal/kg). There were also reductions (p<0.05) in both 22‐hour and dark period RQs in the "non‐eaters" in comparison to those who were "eaters". Monkeys were also classified as "lean" (modified BMI < 25) or "obese" (modified BMI > 30). The obese group had lower EE (p<0.05) during each time period and over the entire 22‐hours (p<0.05), in comparison to their lean counterparts.

Conclusion

There is an association between a reduction of energy expenditure and a gain in body weight.
Prevention of DNA damage by polyphenol‐rich food
Authors: Briviba Karlis , Bub Achim , Watzl Bernhard , Rechkemmer Gerhard , Barth Stephan W
Published: June 2021

Epidemiological and experimental data suggest that an increased intake of plant food rich in polyphenols can reduce the risk of cancers. DNA damage is an important step in the initiation of carcinogenesis. Here we report data on the effect of polyphenol rich food such as apples and apple juice on DNA damage in colon epithelial cells from animal studies and in peripheral blood lymphocytes (PBL) from human intervention studies.

1,2‐Dimethylhydrazine‐induced DNA strand breaks in colon epithelial cells in rats were significantly inhibited by consumption of cloudy and clear apple juice. In a human intervention study, consumption of 1 kg apples caused no changes in endogenous DNA strand breaks, in Fpg‐sensitive sites (oxidative purine damage) or in the capacity to protect DNA against damage induced by hydrogen peroxide in PBL. But, a statistically significant decrease in the levels of endonuclease III sensitive sites (oxidative pyrimidine damage) and an increase in capacity to protect DNA against strand breaks induced by iron chloride were identified. In contrast to the effects of apples, a human intervention with 2, 5 or 8 servings of fruit and vegetables/day had no effect on DNA damage in PBL. Thus, we have identified apples as another fruit to add to the already known types of fruit (kiwi) and vegetables (tomato) with antigenotoxic properties and partly characterized their antigenotoxic mechanism in humans.

Supported by the Deutsche Forschungsgemeinschaft and the Federal Ministry of Research and Education, Germany
Pharmacoequivalence of Enoxaparin and Contaminated Enoxaparin
Authors: Kuziej Jacqueline , Jeske Walter , Hoppensteadt Debra , Litinas Evangelos , McGeehan Elizabeth , Fareed Jawed , Kennedy Richard H.
Published: June 2021

The use of heparin contaminated with oversulfated chondroitin sulfate (OSCS) was associated with adverse reactions. Some batches of enoxaparin also contained low levels of OSCS. Studies were performed to address the bioequivalence of enoxaparin and its contaminated version. Contaminant‐free (CFE) and contaminated enoxaparin (CCE) were administered subcutaneously to rats. Blood pressure and heart rate were measured 90 min. post‐administration, followed by jugular vein clamping model 120 min. post‐administration. Blood was collected to measure anticoagulant and anti‐protease effects. No differences in blood pressure or heart rate were observed between the two groups. Compared to saline treated rats (3.5±0.5 clampings), CCE and CFE treated animals required more clampings to induce thrombosis (4.8±0.7 and 5.0±0.6, respectively; p=0.001 vs. saline). The aPTT was slightly elevated in both CFE and CCE treated groups (CFE: 36.8±18.6 sec; CCE: 30.5±10.9 sec vs. saline: 26.7±3.9 sec). Anti‐Xa activity was significantly higher with CFE (84.4±1.5% inhibition) compared to CCE (80.5±2.9 % inhibition; p=0.026) while anti‐IIa activity was comparable (37.1±22.0 and 30.6±17.9 % inhibition). At <5%, OSCS does not impact the anti‐thrombotic effects enoxaparin. The impact of repeated administration of contaminated enoxaparins and long‐term pharmacodynamic and immunogenic effects needs to be explored further.
The role of MMP2 in persistent inflammation in Chronic Obstructive Pulmonary Disease (COPD)
Authors: Gosselink John , Hayashi Shizu , Elliott W Mark , Xing Li , Chan Becky , Wright Claire , Pare Peter , Sin Don , Pierce John , Pierce Richard , Patterson Alex , Cooper Joel , Hogg James
Published: June 2021

Rationale

MMP2 modifies chemokines in ways that allow them to continue to bind to their receptors but fail to activate them (Science. 2000, 289:1202‐6).

Objective

To investigate the role of MMP2 expression and activity in the development of the persistent inflammatory process associated with progression of COPD.

Methods

The expression of MMP2 was measured by Taqman quantitative PCR in 136 paired samples of small bronchioles and surrounding lung tissue separated by laser capture microdissection from 63 patients in all 4 categories of COPD severity. MMP2 mRNA expression was compared to forced expiratory volume in one second (FEV1) by linear regression analysis. As well, MMP2 activity was measured in 11 of these COPD patients' lungs by gelatin zymography.

Results

MMP2 expression and activity decreased in both the small airways and surrounding lung tissue with disease progression in COPD.

Conclusions

This sharp reduction of MMP2 expression and activity may contribute to the persistence of the inflammatory process in COPD by reducing the proteolytic modification of chemokines that normally blocks chemokine receptors to reduce the inflammatory response.

Supported by grants NIH‐HL‐P50 084922 and CIHR MOP‐82815
Immunohistochemical Examination of Complex Vascular Lesions in the Lungs of Domestic Fowl Selected for Susceptibility to Pulmonary Arterial Hypertension
Authors: Hamal Krishna R. , Wideman R. , Cross D. , Rhoads D. , Anthony N. , Erf G.
Published: June 2021

Pulmonary arterial hypertension (PAH) is a human disease in which vascular remodeling can lead to the formation of plexiform lesions in terminal pulmonary arterioles. Various molecules are expressed by the diverse cell types within human plexiform lesions, including smooth muscle actin (SMA), von Willebrand factor (VWF), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), collagen, and immune cells such as macrophages and CD8+ cells. Plexiform lesions rarely develop in the lungs of the laboratory mammals, but plexiform‐like complex vascular lesions (CVL) do develop spontaneously in the lungs of chickens from a PAH‐susceptible line. This study examined the expression of various molecules by the cells within CVL in chicken lungs. Paraformaldehyde fixed, paraffin embedded sections of lungs from 8‐ to 24‐week old male and female chickens were stained immunohistochemically using monoclonal or polyclonal antibodies. Cells within chicken CVL exhibited positive staining for SMA, VWF, VEGF, VEGFR2, collagen type III, macrophage and CD8 molecules. Foam‐like giant cells within the CVL stained positively as macrophages, whereas positive staining for VWF was detected in tufts of intimal cells and in the capillary endothelium of CVL. Immunohistochemical staining patterns within the CVL of PAH‐susceptible chickens combined with the close histological resemblance of CVL to plexiform lesions of human PAH patients further validates chickens from our PAH‐susceptible line as an excellent animal model of spontaneous plexogenic arteriopathy. Supported by NIH grant 1R15HL092517.
Chronic fluoxetine decreases elevated left ventricular 5‐HT receptor density in rats with congestive heart failure.
Authors: Henze Marcus , Holmes Earle , Battaglia George , Scrogin Karie E
Published: June 2021

Chronic fluoxetine (FLX, 20 mg/kg, s.c.) reduces cardiac β‐adrenergic receptor (βAR) function in rats with congestive heart failure (CHF) and controls. To determine if FLX reduces βAR density or alters 5‐HT receptor (5‐HTR) density, 125I‐cyanopindolol (ICYP, 42 pM) binding was determined in left ventricular tissue of real‐ or sham coronary artery‐ligated rats, given daily FLX or vehicle for 5 wks. ICYP binding was assessed in the absence and presence of 1 μM 5‐HT (to determine 5‐HTR density), 1 μM CGP 20712A (to determine β2 receptor density), and 10 μM propranolol (for non‐specific binding). Ligated rats developed CHF (left ventricular end‐diastolic pressure: 29.7 ± 2.7 vs. 6.5 ± 2.1 mmHg, P<0.01). Serum fluoxetine and norfluoxetine levels did not differ between drug‐treated groups (278 ± 65 vs. 225 ± 31 ng/ml, 1,518 ± 176 vs. 1,440 ± 117 ng/ml, respectively). β1 and β2 receptor binding did not differ between groups. 5‐HTR density was increased in CHF compared to sham rats (2.99 ± 1.12 vs. 0.63 ± 0.27 fmol/mg, P<0.01) and decreased following chronic FLX treatment (2.99 ± 1.12 vs. 0.59 ± 0.43 fmol/mg, P<0.05). FLX reduces 5‐HTR‐, but not β‐receptor density in CHF rats. Supported by AHA 507025.
Metformin Improves Endothelial Function in Obese Rats
Authors: Lobato Nubia Souza , Akamine Eliana Hiromi , Filgueira Fernando Paranaiba , Tostes Rita de Cássia , Carvalho Maria Helena Catelli , Fortes Zuleica Bruno
Published: June 2021

Aim

Obesity is associated with insulin resistance and vascular alterations. Since metformin (MET) improves insulin sensitivity and vascular dysfunction in diabetes, we evaluated the effects of MET on vascular reactivity in monosodium glutamate (MSG) obese rats. Methods and

Results

Wistar rats received MSG from day 2 to day 6 after birth for obesity induction. MSG rats (18 weeks) presented higher Lee index, fat accumulation, dyslipidemia and insulin resistance. MET (300mg/kg/day, 15 days) had beneficial effects on these parameters. In the mesenteric arteriolar bed the response to norepinephrine, enhanced in MSG rats, was corrected by MET. This correction was reverted by L‐NAME and tetraethylammonium (TEA), but not by indomethacin. The response to acetylcholine (ACh), reduced in MSG rats, was corrected by MET. L‐NAME, indomethacin or TEA did not alter this correction. The vasodilation to sodium nitroprusside was enhanced by MET. Althought MET did not alter the reduced nitric oxide (NO) production, it reverted the reduced PGI2/TXA2 release and the enhanced reactive oxygen species (ROS) generation in MSG rats.

Conclusion

MET restores vascular dysfunction in MSG rats by reducing ROS generation, modulating membrane hyperpolarizating and reestablishing the balanced release of prostanoids. The hyperresponsiveness of the smooth muscle to NO with MET treatment might contribute to its beneficial effects. FAPESP
Kaempferol Attenuates the Highly Reducing Sugar‐triggered Inhibition of Osteoblast Differentiation
Authors: Kim Gun‐Hee , Choi Eun‐Mi
Published: June 2021

Type 1 diabetes in humans has as one of its complications inadequate bone formation, resulting in osteopenia and delayed fracture healing. Reducing sugars produce reactive oxygen species through autoxidation and protein glycosylation. 2‐Deoxy‐D‐ribose (dRib) is a reducing sugar with high reactivity. Kaempferol is a natural flavonoid which has been isolated from grapefruit and other plant sources. To determine if kaempferol could influence osteoblast dysfunction induced by dRib mouse osteoblasts, MC3T3‐E1 cells, was treated with 30 mM dRib and kaempferol (10−9‐10−5 M) and markers of osteoblast function and lipid peroxidation were examined. Kaempferol (10−9‐10−5 M) significantly increased the growth of MC3T3‐E1 cells in the presence of 30 mM dRib (p<0.05). Kaempferol caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells. We then examined the effect of kaempferol on the production of osteoprotegerin and malondialdehyde (MDA) in osteoblasts. Treatment with kaempferol (10−9 and 10−5 M) increased osteoprotegerin secretion in osteoblastic cells. Moreover, kaempferol (10−9 and 10−5 M) decreased MDA contents of osteoblastic MC3T3‐E1 cells in the presence of 30 mM dRib. Taken together, these results demonstrate that kaempferol inhibits dRib‐induced damage and may be useful in the treatment of diabetes related bone diseases.
Antiarrhythmic drug‐induced internalization of the atrial specific K+ channel, Kv1.5
Authors: Schumacher Sarah Marie , McEwen Dyke P , Zhang Lian , Arendt Kristin L , Van Genderen Kristin M , Martens Jeffrey R
Published: June 2021

Conventional antiarrhythmic drugs target the ion permeability of channels, but increasing evidence suggests that functional ion channel density can also be modified pharmacologically. Kv1.5 mediates the ultrarapid potassium current (IKur) that controls atrial action potential duration. Given the atrial‐specific expression of Kv1.5 and its alterations in human atrial fibrillation, significant effort has been made to identify novel channel blockers. In this study, treatment of HL‐1 atrial myocytes expressing Kv1.5‐GFP with the class I antiarrhythmic agent quinidine, resulted in a dose‐, time‐, and temperature‐dependent internalization of Kv1.5, concomitant to channel block. Channel internalization was subunit‐dependent, stereospecific, activity‐independent, and blocked by pharmacologic disruption of the endocytic machinery. Pore block and channel internalization partially overlap in the structural requirements for drug binding. Surprisingly, quinidine‐induced endocytosis was calcium‐dependent and therefore unrecognized by previous biophysical studies focused on isolating channel‐drug interactions. Together, these data reveal a novel mechanism of antiarrhythmic drug action and highlight the possibility for new agents that selectively modulate the stability of channel protein in the membrane as a novel approach for treating cardiac arrhythmias. Supported by NIH HL0270973, JRM.
TRPA1 Antagonists Block the Noxious Effects of Toxic Industrial Isocyanates and Tear Gases
Authors: Bessac Bret F. , Sivula Michael , von Hehn Chrisitan A. , Caceres Ana I. , Escalera Jasmine , Jordt Sven E.
Published: June 2021

The release of the methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation and edema. Similar responses are elicited by chemicals used as tear gases. Despite of frequent exposures the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective countermeasures. We use Ca2+ imaging and electrophysiology to show that the noxious effects of isocyanates and all major tear gas agents are caused by activation of Ca2+ influx and membrane currents in mustard oil‐sensitive sensory neurons. These responses are mediated by Transient Receptor Potential Ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate‐ and tear gas‐induced nocifensive behavior following both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. TRPA1 antagonists can prevent and alleviate the adverse health effects of exposures to a wide range of toxic noxious chemicals.
Reference moist smokeless tobacco‐induced oxidative stress in human oral keratinocyte cell lines leads to cell death via JNK and p38 MAPK signaling
Authors: Mitchell Clint , Piper John , Joyce Andrew , Fariss Marc , McKallip Robert
Published: June 2021

The use of moist smokeless tobacco products is associated with acute oral injuries such as leukoplakia. Unfortunately, there is a limited understanding of the mechanisms and signaling pathways associated with these injuries. The present studies investigate the effects of a Reference 1S3 moist smokeless tobacco extract (STE) on oral injury, oxidative stress and MAPK signaling in multiple human oral keratinocyte cell lines utilizing cytospin slide analysis to observe alterations in cell morphology, DCF‐DA fluorescence, and western blotting techniques. STE caused dose‐dependent keratinocyte cell death within 30 minutes to 3 h of exposure. STE enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 or p38 MAPK, but not ERK1/2, suppressed STE‐induced cell death. STE promoted the generation of reactive oxygen species (ROS). The anti‐oxidants, Trolox and L‐ascorbic acid, suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Thus, reference STE leads to cell death in human oral keratinocyte cell lines in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.

Research supported by Altria Client Services, 601 East Jackson St. Richmond, VA 23219 U.S.A.
Pharmacologic activation of the tumor suppressor PP2A as a potential therapy for treating Jak2 V617F‐driven myeloproliferative disorders
Authors: Oaks Joshua , Santhanam Ramasamy , Briesewitz Roger , Perrotti Danilo
Published: June 2021

Polycythemia vera (PV) is a myeloproliferative disorder caused by the V617F mutation in the inhibitory JH2 domain of the signaling kinase Jak2 that, reportedly, is tightly regulated by the tumor suppressor phosphatase PP2A. Here we show that Jak2V617F inactivates PP2A in a dose/kinase‐dependent manner (98% inhibition; P< 0.05) in Ba/F3 and TF‐1cells hematopoietic precursor cell lines. Moreover, treatment with 600nM Jak inhibitor I totally rescued PP2A activity. In these cells, knockdown of the endogenous PP2A inhibitor SET restored PP2A activity up to 93% (P<0.01), suggesting that SET is necessary for suppression of PP2A activity. To evaluate the potential benefit of PP2A reactivation in PV, Jak2V617F‐expressing Ba/F3 and lineage‐negative mouse bone marrow cells were treated with the PP2A activator FTY720 at 2.5µM. FTY720 restored PP2A activity to 75% and 100% in Jak2V617F‐expressing Ba/f3 and bone marrow cells (P< 0.01), respectively. Also, FTY720 reduced the clonogenic potential of Ba/f3‐Jak2V617F cells by 84%. These results suggest that Jak2V617F is regulated by PP2A and that pharmacologic activation of PP2A represents a potential avenue for treatment of Jak2 V617F‐driven myeloproliferative disorders.
Phospholipase C beta 1b is the PLC splice variant that selectively mediates α1‐receptor and Gq‐coupled hypertrophy in cardiomyocytes
Authors: Filtz Theresa Marie , Grubb David Robert , Woodcock Elizabeth Ann
Published: June 2021

Activation of the heterotrimeric G protein Gq in cardiomyocytes leads to pathological hypertrophy and apoptosis in vivo and in vitro. As the immediate downstream effector of Gq, phospholipase Cβ (PLCβ) may mediate these responses. Neonatal rat cardiomyocytes (NRVM) express the two splice variants of PLCβ1, PLCβ1a (150 kDa) and PLCβ1b (140 kDa), which differ only in their extreme C‐termini. PLCβ1a is localized in the cytoplasm of NRVM whereas PLCβ1b targets to the sarcolemma through its unique C terminal domain. Adenoviral over‐expression of PLCβ1b in NRVM caused substantial hypertrophy followed by apoptosis; whereas over‐expression of PLCβ1a did not. Adenovirus expression in NRVM of an inhibitory mini‐gene incorporating the entire unique C‐terminal sequences of PLCβ1b inhibited PLC activity and completely suppressed hypertrophy induced with phenylephrine/propanolol or Gq over‐expression. This study indicates a major role for PLCβ1b in Gq‐mediated pathology and suggests that PLCβ1b but not PLCβ1a is responsible for Gq responses in NRVM. The PLCβ1b/sarcolemmal interface provides a potentially selective target for blocking Gq‐mediated pathology. This work was sponsored by the NH&MRC (Australia).
Molecular mechanisms associated with peptidergic control of adrenergic function
Authors: Wong Dona Lee , Tai T.C. , Wong‐Faull David C. , Claycomb Robert , Aborn Jennifer H.
Published: June 2021

Phenylethanolamine N‐methyltransferase (PNMT), which produces epinephrine, is a marker of adrenergic function. While pituitary adenylate cyclase activating polypeptide (PACAP), independently and cooperatively with NGF, activates PNMT promoter‐driven and endogenous PNMT gene expression in PC12 cells, molecular mechanisms remain unknown. Transfection assays in PC12 cells and PKA‐deficient and PLCγ1‐deficient PC12 cells using PNMT promoter‐luciferase gene constructs and signaling inhibitors showed that PLCγl and cAMP‐dependent PKA signaling are critical for PACAP activation and PI3K, PKC, ERK1/2 MAPK, p38 MAPK downstream. The inhibitors also abrogated PACAPergic induction of endogenous PNMT. Western analysis of nuclear protein from PACAP‐treated PC12 cells showed that Egr‐1 and AP‐2 underlie PACAP responses. Results with nested deletion or site‐directed mutant constructs support this possibility. Exposure of PACAP‐treated transfected and untransfected PC12 cells to histone deacetylase (HDAC) inhibitors, Na butyrate or Trichostatine A, incrementally activated the PNMT promoter and endogenous PNMT. Findings indicate that PACAP transcriptionally activates the PNMT gene with HDAC being limiting. Presence of long and short forms of PNMT mRNA further suggest post‐transcriptional regulation by PACAP. Support: Spunk Fund, Inc., Sobel‐Keller Fund and McLean Hospital.
Transcriptional Regulation of the Human Neutral Ceramidase Gene
Authors: O'Neill Sean M , Kester Mark
Published: June 2021

Many forms of cellular stress cause an elevation of endogenous, apoptotic, ceramide (Cer) levels. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine‐1‐phosphate. To further define the physiological regulation of neutral CDase (nCDase), we used a luciferase reporter analysis system to define the proximal promoter of the nCDase gene and to demonstrate that serum can activate this proximal promoter, which corresponded to a serum‐induced increase in human nCDase mRNA expression. Computational analysis identified candidate transcriptional response elements (TRE), and electrophoretic mobility shift assays (EMSA) demonstrated that these TRE bind their respective transcription factors, including NF‐Y, AP‐2, Oct‐1, GATA, and serum‐activated AP‐1. Mutagenic analyses of the TRE revealed that these sites, including the AP‐1 cis‐element, regulate the promoter and are necessary for maximal activity of the proximal promoter. Moreover, siRNA knockdown of the AP‐1 subunit, c‐Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c‐Jun overexpression was sufficient to increase its activity. Thus, the transcription factor AP‐1 can directly regulate the nCDase promoter. Taken together, our findings suggest that c‐Jun/Ap‐1 signaling may regulate human nCDase gene transcription. These studies were funded by NIH HL076789 to MK.
Prenatal and Postnatal Depletion of L‐Aromatic Aminoacid decarboxylase (LAAD) by MPTP as a Sensitization and Precipitation Model for Parkinson's disease
Authors: Muthian Gladson , Mackey Veronica , King Jennifer , Charlton Clivel George
Published: June 2021

Parkinson's disease (PD) is due to the degeneration of dopamine (DA) neurons and loss of DA and the dopaminergic enzymes, including L‐aromatic aminoacid decarboxylase (LAAD) in the nigrostriatal (NS) system. In our studies we test the hypothesis that timed exposure of fetuses to toxins sensitizes the NS DA neurons and that later challenges further harm the vulnerable neurons, resulting in PD. Thus, a sensitization stage and a precipitating stage underlie idiopathic PD. For the 1st/sensitization stage we exposed C57B/L mouse fetuses to 10mg/kg/dy 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) during gestation day 8‐12 to target the emerging NS DA neurons. For the 2nd/precipitating stage the adult offspring were challenged with 10, 20 or 30 mg/kg/dy MPTP for 7 days. At 7dy post‐MPTP the analysis of LAAD and DA and its metabolites were performed. The results show that prenatal MPTP has a long lasting effect and may be specific for the NS system, since in the 3mths old offspring striatal and brainstem, but not cortical, LAAD expressions were reduced. Postnatal MPTP is additive and further decreases LAAD in a dose‐dependent manner. Prenatal MPTP did not significantly reduce cortical DA, as compared to PBS but it sensitizes DA to postnatal MPTP in different concentration. The outcome demonstrates that idiopathic PD may have a fetal basis and that chemical and/or functional stress and age‐related changes precipitate PD symptoms by causing further harm. Supported by NIH RO1NS041674 and NIH R21NS049623.