bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Trim32‐catalyzed polyubiquitin chain formation
Authors: Streich Frederick C , Haas Arthur L
Published: June 2021

Trim32, a member of the Trim superfamily of ubiquitin ligases defined by a conserved tripartite RING‐β box‐coiled coil motif, was originally discovered as an HIV‐I Tat interacting protein and has been implicated in limb‐girdle muscular dystrophy, Bardet‐Biedl syndrome, and displays anti‐apoptotic activity associated with carcinoma. Trim32‐catalyzed polyubiquitination of target proteins is a critical mechanism by which proteins are marked for 26S proteasome‐dependent degradation; however, little is known regarding the mechanism of conjugation or chain elongation among members of this superfamily. Kinetic studies show, contrary to the literature, that the Ubc5 but not UbcH6 family is the cognate E2 supporting Trim32 conjugation. Trim32 catalyzes a slow rate of free polyubiquitin chain formation in the absence of substrate and demonstrates cooperativity with respect to [Ubc5A]o (n=3.4), suggesting four tightly coupled E2 binding sites. Cooperativity requires subunit association through the βbox‐coiled coil motif since the RING domain alone is incapable of polyubiquitin chain assembly. Ring domain dimerization through N‐terminal GST‐mediated association reconstitutes ubiquitin chain formation. Characterizing the mechanism of polyubiquitin chain formation will help describe why mutants of Trim32 and other Trim proteins are associated with debilitating diseases. [Funded by GM340092 to ALH]
Rapid Freezing Quench of Transient Conformations of Folding Proteins for Solid State NMR Studies
Authors: Hu Kan‐Nian , Yau Wai‐Ming , Tycko Robert
Published: June 2021

Rapid freezing of samples brought new applications of biomolecular solid state nuclear magnetic resonance (SSNMR) to the structural characterization of kinetically trapped intermediates in protein folding. A frozen protein solution from an elevated temperature up to 90 °C was transferred to an SSNMR spectrometer, where structural measurements were carried out at < ‐120 °C for many days. We initially studied a small helical protein HP35 (35‐residue villin headpiece subdomain) with high stability (70 °C unfolding temperature) and a rapid folding rate(10 μs time scale). A 13C‐labeled HP35 solution in glycerol/water was cooled from 90 to ‐120 °C in ~10 μs by spraying the hot solution through a 20μm‐diameter nozzle into a cold isopentane bath, generating slurry of frozen protein solution with ~10 μm particle sizes. 13C NMR spectra show both folded and unfolded protein components in approximately equal proportions for some sites and indicate that the HP35 folding process is not cooperative for several labeled amino acid residues. Moreover, the tertiary contacts and backbone ordering do not form simultaneously through folding.
GDP‐mannose transporter contributes to polarity establishment in the filamentous fungus Aspergillus nidulans
Authors: Johnson Laura , Gordon Barbara , Groover Chassidy , Martin Stuart , Hill Terry , Loprete Darlene , Jackson‐Hayes Loretta
Published: June 2021

GDP‐mannose transporters (Gmt) carry nucleotide sugars from the cytosol across the Golgi apparatus membrane in various eukaryotic organisms including plants and a variety of fungi. Some fungal species such as the yeast Saccharomyces cerevisiae express only one Gmt, while others including A. nidulans express paralogues (GmtA and GmtB) whose individual roles have not been elucidated. As in S. cerevisiae GmtA is localized within the Golgi apparatus which in A. nidulans consists of widely separated cisternae, in contrast to the stacked cisternae of animals and plants. Here we show that GmtB localization is congruent with GmtA in mature hyphae. Real‐time reverse transcriptase PCR shows a gradual decrease in GmtA expression during the first 12 hours of germination while GmtB expression remains high and unchanged during this period. Depletion of GmtB by the glucose repressible alcA promoter causes abnormalities in polarized growth, a developmental event that is initiated during the early hours of germination. Taken together these observations suggest that GmtB plays an important role in establishment of polarized growth. Although GmtA and GmtB reside within the same Golgi compartments they appear to perform independent functions at different developmental stages.

Funded by Research Corporation, the Merck/AAAS Undergraduate Science Research Program and NSF RUI
A Biochemical Driver for Living Systems: the Proton Pump
Authors: Haines Thomas H.
Published: June 2021

All living membranes (as apposed to myelin, a non‐living membrane) pump protons or other ions. The introduction of the sources of energy upon which living cells rely, enter cells via ion, commonly proton, pumping to make ATP. These energy sources consist of either redox energy or photons. Heterotrophic cells may take in energy in the form of glucose or other covalent bond sources, but the energy is converted into NADH or its equivalent by metabolism. The reductive product is then transduced via proton pumping to ATP production. In eukaryotes some of that ATP may be used thereafter to pump protons in other, internal cellular membranes.

Thermodynamically, living cells characteristically use energy to create order. The equations that explain this apparent contradiction of the 2nd Law of Thermodynamics were best derived by Ilya Prigogene and Ephraim Katchalsky who established that such activities must be near‐to‐equilibrium and must be local within a larger surrounding environment that increases the entropy. Prigogene showed that although local, near‐to‐equilibrium systems may be interlocked. Thus, for example, if the act of proton pumping were reversibly connected to ATP synthesis, the ATP could then be used as a key component of other near‐to‐equilibrium systems such as metabolism and/or cytoskeletal activities.

I propose a specific mechanism for the act of proton pumping that would qualify as a near‐to‐equilibrium system. It embodies a motion perpendicular to the plane of the membrane for the pumping of protons. The mechanism is both cooperative and synchronized. Each transport event results in a compaction of the protein across the membrane provoking a neighboring pump to expand and visa versa. The mechanism implies that pumps be dimers or multimers in living membranes although they could pump individually in reconstituted in bilayers. It is the nature of the pumping mechanism that it can be restarted with Brownian motion if it falters.
Separate Regions of Glucocorticoid Receptor, Coactivator TIF2, and Comodulator STAMP Modify Different Parameters of GR‐mediated Gene Induction
Authors: Awasthi Smita , Simons S. Stoney
Published: June 2021

Coregulators, such as TIF2, and comodulators, such as STAMP, modify several aspects of glucocorticoid receptor (GR)‐mediated gene induction. Varying the levels of these factors alters not only the total level of transactivation (Amax) but also both the position of the dose‐response curve (e.g., EC50) for agonists and the amount of partial agonistic activity of antagonists (as a percent of full agonist activity under the same conditions). Our lab has found that TIF2 and STAMP appear to form a complex with GR that results in more than additive changes in Amax, EC50, and percent partial agonist activity for GR‐inducible genes. Using gene activation, mammalian two‐hybrid, and co‐immunoprecipitation (co‐IP) assays with truncated proteins; we find that selected regions of each protein (GR, TIF2, and STAMP) are required in the ternary complex to modify some, but not all, induction properties. Modulation of EC50 requires the most domains of each protein. Smaller fragments are sufficient to modify the Amax and/or percent partial agonist activity. Even less is required to form a complex. The data suggest that the pathways controlling Amax, EC50, and percent partial agonist activity of GR transactivation are physically separable and may utilize different transcriptional cofactors. Thus, greater physiological control of glucocorticoid action may be possible via agents that modify individual induction parameters.

Source of research support: NIH Intramural Program
Regulation of HRas signaling by Rabex‐5‐mediated ubiquitination
Authors: Xu Lizhong , Lubkov Veronica , Taylor Laura , Bar‐Sagi Dafna
Published: June 2021

Mammalian cells express three isoforms of Ras proteins, HRas, NRas and KRas4B (referred to as KRas hereafter). Each isoform plays an important role in controlling a wide spectrum of fundamental cellular processes. We have recently shown that the location‐specific signaling activity of HRas and NRas but not KRas is regulated by ubiquitination. The objective of this study is to identify the molecular mechanism by which Ras ubiquitination is regulated. Using the in vivo and in vitro ubiquitination assays along with RNA interference technology, we have found that the E3 ligase Rabex‐5 (also known as RabGEF1), but not its ligase‐deficient counterpart (Y25A/Y26A), is necessary and sufficient to catalyze Ras ubiquitination. Rabex‐5‐mediated HRas ubiquitination promotes its endosomal localization and leads to the suppression of ERK activation. Furthermore, the Ras effector RIN1 enhances HRas ubiquitination through its Rab5 GEF activity in a Rabex‐5 dependent manner. Thus RIN1 and Rabex‐5‐dependent Ras ubiquitination represents a new modality for the regulation of Ras compartmentalization and signaling.

This work was supported by research grants from National Institutes of Health to D.B.‐S. (CA55360 and GM078266).
NGF activates NFAT‐dependent transcription via the PI3K‐Akt‐GSK3β pathway in rat sensory neurons
Authors: Kim Man Su , Usachev Yuriy M
Published: June 2021

Nuclear Factor of Activated T‐cell (NFAT) regulates a number of neuronal functions, such as axonal growth, synapse formation and neuronal survival. In addition to electrically mediated Ca2+ signal, gene transcription by NFAT is modulated by nerve growth factor (NGF) via poorly understood mechanisms. In rat sensory neurons, we found that NGF potentiated NFAT‐dependent transcription of a luciferase reporter gene induced by mild depolarization. This effect was diminished by TrkA receptor inhibition. We hypothesized that NGF potentiates NFAT‐mediated transcription by decreasing GSK3β activity via the phosphoinositol‐3‐kinase (PI3K)/Akt pathway downstream of TrkA activation. We found that pharmacological inhibition of PI3K and Akt significantly reduced NGF‐dependent facilitation of NFAT‐mediated transcription. While GSK3β inhibitor increased NFAT activity, overexpression of constitutively active GSK3β blocked the NGF‐dependent facilitation of NFAT activation. Moreover, confocal microscopy revealed that GFP‐NFAT nuclear translocation was increased in the presence of NGF. In summary, our data suggest that NGF utilizes the PI3K‐Akt‐GSK3β axis to facilitate NFAT‐mediated transcription triggered by electrical activity. Thus, NFAT may serve as an integrator of electrical activity and neurotrophin signaling in the regulation of neuronal gene expression. This work was supported by AHA.
PDE3A may regulate myocardial Ca2+ cycling through interacting with SERCA2 in mouse heart
Authors: SHEN WEIXING , SUN JUNHUI , HOCKMAN STEVEN , KHAN FAIYAZ , MANGANIELLO VINCENT
Published: June 2021

Growing evidence indicates that cardiac functions, from contractility to chronic gene expression and cardiac structural remodeling, are regulated by multiple spatially, temporally, and functionally distinct pools of cAMP. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cGMP, regulate the amplitude, duration, and compartmentation of cAMP‐mediated signaling. PDE3 plays an important role in regulating cAMP metabolism in the cardiovascular system. Although PDE3 inhibitors, by raising cAMP, produce acute inotropic and vasodilatory effects, the mechanism for these actions are unclear. In our experiments, we found that PDE3A co‐localizes with SERCA2 and phospholamban during sucrose gradient centrifugation of mouse cardiac membranes. In addition, using LC‐MS/MS analysis of PDE3A immunoprecipitates, we found that PDE3A immunoprecipitated with sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2), which regulates Ca2+ uptake into intracellular stores. Immunoblot and enzyme activity assays also indicated that PDE3A immunoprecipitated with SERCA2, and other signaling molecules. These data suggest that, as a component of a SERCA2‐containing macromolecular complex, PDE3A regulates a discrete cAMP pool important in mediating some effects of SERCA2 on cardiac function. Compared with wild type mice, expression of SERCA2 and phosphorylation of phospholamban at Ser16 were increased in heart tissue from PDE3A‐/‐ mice, generated in our laboratory. Effects of PDE3A on Ca2+ cycling and ventricular function will be investigated in future studies.
Glutamatergic Receptor Activation in the Rostral Ventrolateral Medulla Contributes to the Sympathoexcitatory Response of Hyperinsulinemia
Authors: Bardgett Megan Elyse , Stocker Sean D
Published: June 2021

Hyperinsulinemia increases sympathetic nerve activity (SNA) and has been linked to cardiovascular morbidity in obesity. Many sympathoexcitatory responses are mediated by glutamatergic receptor activation of spinally projecting neurons in the RVLM. Therefore, we hypothesized that ionotropic glutamate receptors in the RVLM contribute to elevated SNA during hyperinsulinemia. Hyperinsulinemic‐euglycemic clamps were performed in urethane‐anesthetized, male Sprague‐Dawley rats (270‐350g, n=8) by IV infusion of insulin (7.5 mU/kg/min) and 50% dextrose (.25‐.75 ml/hr) for 120 min. After 90 min, hyperinsulinemic clamps significantly increased plasma insulin (2.5±0.9 to 12.2±3.9 ng/ml, P<0.05), elevated lumbar (125±18%, P<0.01) and renal SNA (48±17%, P<0.05) without any significant change in arterial blood pressure (0 min: 125±3 vs 90 min: 118±4 mmHg) or plasma glucose levels (0 min: 107±9 vs 90 min: 111±10 mg/dl). RVLM injection of the glutamate receptor antagonist kynurenic acid (5 nmol/60 nL/ side, n=4) vs saline (n=4) significantly reduced lumbar SNA (‐41±1% vs 3±1%, P<0.01) but did not significantly alter renal SNA (27±14% vs 0±3%) or arterial blood pressure (1±2 vs ‐1±1 mmHg). These findings indicate that hyperinsulinemia increases lumbar SNA, in part, through glutamatergic receptor activation in the RVLM.

Supported by NIH HL090826, AHA 0630202N & 0815372D
Oxidative Stress Induces Redistribution of Claudins in Caco‐2 Cell Monolayers by a Tyrosine Kinase‐Dependent Mechanism
Authors: Seth Ankur , Samak Geetha , Rao R.K.
Published: June 2021

Oxidative stress promotes colorectal cancer and tumor metastasis. Destabilization of tight junctions (TJs) plays a key role in the process of tumor metastasis. Claudins are involved in assembly and maintenance of TJs. In the present study, we evaluated the effect of H2O2 on Tyr‐phosphorylation and distribution of claudins in Caco‐2 cells. Caco‐2 cell monolayers were incubated with a Tyr‐kinase inhibitor, genistein (100 μM) or Src kinase inhibitor, PP2 (3 μM) for 60 min prior to incubation with H2O2 (20 μM) for 1‐3 hours. TJ integrity was evaluated by measuring inulin permeability. Distribution of claudins (Cln‐1, Cln‐3, Cln‐4 & Cln‐5) and ZO‐1 was analyzed by immuofluorescence confocal microscopy. Triton‐insoluble and soluble fractions were immunoblotted for different claudins. Tyr‐phosphorylation of claudins was determined by immunoprecipitation and immunoblot analysis. Results show that H2O2 induces a redistribution of Cln‐1, Cln‐3, Cln‐4 and Cln‐5 from the intercellular junctions into the intracellular compartments, which was attenuated by genistein or PP2. H2O2 induced a significant reduction of Cln‐3 in the Triton‐insoluble fraction, while increasing its level in Triton‐soluble fraction. On the other hand, Cln‐1, Cln‐2 and Cln‐4 levels in Triton‐insoluble fractions were increased by H2O2; while Cln‐5 level remained unchanged. H2O2 increased Tyr‐phosphorylation of Cln‐3, while it reduced the phosphorylation of Cln‐4. These results indicate that oxidative stress induces Tyr‐phosphorylation and redistribution of claudins in Caco‐2 cell monolayers by an Src kinase‐dependent mechanism.
Survivin is a key factor in the differential susceptibility of gastric endothelial and epithelial cells to alcohol‐induced injury
Authors: Jones Michael K. , Padilla Oscar R. , Zhu Ercheng , Ngov Melisa
Published: June 2021

We previously demonstrated that the anti‐apoptosis protein, survivin, plays a protective role against alcohol‐induced gastric injury. Since the endothelia is a primary target of alcohol‐induced gastric damage, we investigated whether survivin expression is a key factor in the greater susceptibility of gastric endothelial vs. epithelial cells to alcohol‐induced injury. Here, we demonstrate that rat gastric epithelial cells expressed 8‐fold (P<0.01) greater survivin levels vs. rat gastric endothelial cells. Survivin expression correlated (r=0.84, P<0.05) with resistance of gastric epithelial vs. endothelial cells to both alcohol‐induced cell damage (20 ± 3% vs. 65 ± 7%, P<0.05) and alcohol‐induced apoptosis (14 ± 2% vs. 32 ± 4%, P<0.05). Forced overexpression of survivin in gastric endothelial cells significantly increased resistance to both alcohol‐induced damage and apoptosis. Moreover, overexpression of a threonine‐34 to glutamate mutant survivin construct rendered gastric endothelial cells significantly more resistant to alcohol‐induced damage and apoptosis vs. non‐transfected gastric epithelial cells (P<0.05). These findings indicate that disparate survivin expression levels can explain the discrepancy between gastric epithelial and endothelial cell susceptibility to alcohol‐induced injury; and, survivin phosphorylation is important in mediating gastric mucosal protection.

NIH R01AA14946
Oxidative stress mediates activation of NF‐κB and contributes to pathogenesis of streptozotocin‐induced diabetes
Authors: Kang Yu‐Ming , Qin Da‐Nian , Elks Carrie , Francis Joseph
Published: June 2021

Basal neuronal activity in the PVN of diabetic rats is increased; however, the mechanism is not known. We hypothesized that reactive oxygen species (ROS) may activate NF‐κB in cardiovascular regions of the brain and induce downstream synthesis of excitatory elements including pro‐inflammatory cytokines (PICs) in progression of diabetes. Sprague‐Dawley rats were injected with streptozotocin (STZ, 65 mg/kg, ip) to induce diabetes, and treated for 4 weeks with a continuous intracerebroventricular (ICV) infusion of NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC, 5µg/h), or tempol (TEMP, 80µg/hr), a superoxide scavenger, or vehicle. There were higher plasma levels of glucose and PICs (TNF‐α, interleukin (IL)‐1β and IL‐6) and lower insulin in plasma and pancreatic islets in the STZ‐induced diabetic rats compared to control rats. Diabetic rats had high levels of Fra‐LI (an indicator of chronic neuronal activation), PICs, phosphorylated IKKβ (p‐IKKβ), superoxide and gp91phox (a subunit of NAD(P)H oxidase) in the PVN. ICV treatments with PDTC or TEMP for 4 weeks reduced the plasma levels of PICs, expression of Fra‐LI, PICs, p‐IKKβ, superoxide and gp91phox in the PVN of the STZ‐induced diabetic rats. These findings suggest that ROS mediate activation of NF‐κB in the PVN and contribute to pathogenesis of diabetes. Supported by NIH HL‐080544.
Estrogen Receptor β in Female Adipose Tissue: Impact on Metabolic Dysregulation in a Model of Senescence and Estrogen Deficiency
Authors: Tomicek Nanette J. , Lancaster Timothy S. , Korzick Donna H.
Published: June 2021

Background

Obesity and associated metabolic risk are increased following menopause and may be associated with disturbances in estrogen receptor (ER) signaling in adipose depots. However, the role of ER subtypes on increasing adiposity with aging remains uninvestigated.

Purpose

To determine the singular and combined effects of aging and estrogen deficiency on ER levels and to identify dysregulated downstream signaling in adipose tissue of female rats.

Methods

Visceral adipose tissue was isolated from adult and aged female F344 rats (n = 5‐6/group) with ovaries intact or removed (OVX). Protein levels were assessed with western blotting and circulating estradiol through radioimmunoassay.

Results

Rat weight increased significantly with both age and OVX and was greatest in aged OVX. ER β levels increased two‐fold in both aged and aged OVX, while ERα decreased in adult OVX only. PPARγ2 levels were also reduced in aged OVX, in conjunction with a non‐significant increase in adiponectin receptor 1 levels (p = 0.098).

These data suggest, for the first time, that alterations in adipose ERβ levels may contribute to obesity and altered metabolic risk with age‐associated estrogen deficiency. ERβ‐mediated suppression of PPARγ2 may also represent a potential downstream signaling mechanism to exacerbate metabolic risk with post‐menopausal estrogen deficiency via alterations in adiponectin signaling.
Chronic intermittent hypoxia (CIH) disrupts neurovascular coupling and endothelium dependent responses in the mouse cerebral microcirculation
Authors: Iadecola Costantino , Capone Carmen , Coleman Christal , Pickel Virginia , Wang Gang
Published: June 2021

Sleep apnea, which leads to CIH, increases the incidence of stroke, but the mechanism is unknown (AJP 293:R1671, 2007). Brain function depends on a well regulated cerebral blood flow (CBF). The CBF increases induced by neural activity and endothelium‐dependent vasodilation are key factors in CBF regulation, and their dysfunction increases the brain's susceptibility to injury. We hypothesize that CIH exerts its deleterious effects on the brain by altering CBF regulation. Cerebrovascular regulation was examined in mice (n=5/group) subjected to CIH or sham (Sh) treatment for 35 days. Under chloralose‐urethane anesthesia, somatosensory cortex CBF was monitored by a laser‐Doppler probe through a cranial window. CIH elevated mean arterial pressure from 73±2 to 83±3 mmHg (p<0.05) and attenuated (p<0.05) the CBF increase induced by neural activity (produced by whisker stimulation; Sh: 23±1; CIH: 14±1±‐%), and by the endothelium‐dependent vasodilator acetylcholine (10 μM topical; Sh: 22±1; CIH: 13±1%). In contrast, the response to adenosine (100 μM) was not affected (Sh: 23±1; CIH: 25±1%; p>0.05), attesting to preservation of smooth muscle function. We conclude that CIH selectively impairs key homeostatic responses of the cerebral circulation, raising the possibility that CIH‐induced CBF dysfunction promotes cerebrovascular insufficiency and ischemic injury in sleep apnea patients (Supported by HL18974).
Mathematical model of metabolic systems: application and validation by metabolome
Authors: Suematsu Makoto , Kinoshita Ayako Yachie
Published: June 2021

ATP maintenance costs large energy for erythrocytes that mostly depend on glycolysis, although mechanisms for balancing its consumption and synthesis remain unknown. The large‐scale mathematical model of erythrocyte metabolism predicted that uptake of extracellular adenosine into the cells to supply the substrate for ATP synthesis constitutes a major fraction of the consumption: this process causes a decrease in the basal ATP amounts in the cell under normoxia, but not under hypoxia, since the hemoglobin‐dependent O2 sensing activates glycolysis. A shortage of ATP during the adenosine uptake was predicted to be compensated by the hypoxia‐induced glycolytic activation until 20 min after the onset of hypoxia, whereas further lengths of hypoxia did not compensate it. Such a prediction was consistent with the data indicating temporal ATP measurements in human erythrocytes in the presence of adenosine under hypoxia. Based on the model prediction that adenosine kinase and adenylate kinase constitute the major fraction of ATP consumption during the adenosine adsorption, we conducted experiments under the 15N‐labeled adenosine incubation, measuring the mass‐labeled AMP, ADP and ATP in the cells. Results indicated that hypoxia stimulated the uptake of the mass‐labeled adenosine to synthesize the high‐energy adenosine phosphates. When pretreated with CO that inhibits the hypoxia‐induced glycolytic activation, erythrocytes decelerated the salvage of the mass‐labeled adenosine. These results predicted by biosimulation and demonstrated by measurements suggest that hypoxia‐triggered glycolytic activation plays a crucial role in compensating cellular energetics for maintaining adenylate pool through salvaging extracellular adenosine.
Amino Acid Composition and Protein Content of Mature Human Milk from Nine Countries
Authors: Feng Ping , Gao Ming , Holley Timothy , Zhou TianHui , Burgher Anita , Trabulsi Jillian , Pramuk Kathryn , Nazzario Judith
Published: June 2021

This study represents analysis of 220 human milk samples from nine cities in nine countries crossing five continents. Samples were collected from mothers from 18 to 40 years of age with a lactation range from 30 days to 188 days. Amino acids were analyzed using Waters Accq_Tag HPLC method. Total protein was calculated from the nitrogen content multiplied by a protein factor of 6.25. The nitrogen content was analyzed using FP‐528 nitrogen analyzer made by LECO Corporation. True protein was calculated from amino acid analysis and includes protein, free amino acids and peptides. Total protein, true protein, and the amino acid composition are not influenced by mother's age, and are statistically similar among locations, with the exception of samples from Chile, which had higher total protein and true protein. True protein is approximately 76% of total protein, is statistically similar among locations, and is not influenced by mother's age or days of lactation. Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month than in the following four months. The amino acid profiles are statistically similar among locations. These results confirm higher protein and amino acid levels in early lactation and demonstrate consistency in the protein and amino acid composition of human milk across all locations.
Effect of decaffeinated coffee on the serum glucose and insulin responses elicited by oral glucose
Authors: Matusheski Nathan V. , Wolever Thomas M. S. , Black Richard M.
Published: June 2021

A large amount of epidemiologic evidence has emerged indicating an association of coffee consumption with decreased risk for type‐2 diabetes. However, pure caffeine may acutely decrease insulin sensitivity. We conducted a randomized controlled crossover study to determine the effect of an Arabica and Robusta blend decaffeinated coffee (DC), administered either before or concurrently with 75g oral glucose (OGTT), on serum glucose and insulin responses. Ten healthy subjects were studied on 4 separate occasions after overnight fasts. DC was consumed with an OGTT (DC0), or 30min (DC‐30) or 60min (DC‐60) before the OGTT, or not at all (Control). Water was consumed 30min before the OGTT in the control treatment. The incremental area under the curve (AUC) for glucose after DC‐60 was similar to control, but significantly less than that after DC0. There were no differences in insulin response between treatments. An insulin sensitivity index based on fasting glucose and insulin concentrations (HOMAr) at 0 min tended to be less (p=0.052) on DC‐60 compared to DC0. These results suggest that proximity to a meal may influence the effect of decaffeinated coffee on glycemic response. Additional research should examine whether Arabica and Robusta coffee sources possess similar efficacy.
HMGB1/TLR4 axis contributes to A/R‐induced cardiomyocyte apoptosis through potentiating TNFα/JNK pathway
Authors: Xu Xuemei , Yao Yongwei , Kao Raymond , Martin Claudio M , Rui Tao
Published: June 2021

The present study is to explore the role of HMGB1/TLR4 axis in the myocyte apoptosis‐induced by A/R (an in vitro counterpart to I/R).

Methods

cardiomyocytes derived from wild type or TLR4 deficient mice were challenged with an A/R. HMGB1, TNFα expression (Western blot and ELISA), myocyte apoptosis (caspase 3 activity and cell death ELISA) and JNK and NFκB activation (p65 phosphorylation) (Western blot) assessed.

Results

A/R challenge to myocytes resulted in an increase in HMGB1 expression and extracellular release. Inhibition of HMGB1 attenuated the apoptosis and A/R‐induced TNFα production. Treatment of cardiomyocytes with recombinant HMGB1 (1–5 μg/ml) did not lead to myocyte apoptosis and TNFα production. Exogenous TNFα induced a moderate pro‐apoptotic effect on the myocytes; an effect substantially potentiated by HMGB1. TNFα activated both JNK and NFκB. However, degree of TNFα–induced JNK activation was greater than NFκB in the presence of HMGB1. Inhibition of JNK diminished the myocytes apoptosis‐induced by either TNFα or TNFα/HMGB1 cocktail. A/R‐challenge to TLR4 deficient myocytes increased HMGB1 but not apoptosis, TNFα production and JNK activation.

Conclusions

our results indicate myocyte HMGB1/TLR4 axis and TNFα work in concert to promote A/R‐induced myocyte apoptosis via JNK activation. (HSFO NA‐6316).
Glial response during adaptive and maladaptive reactive synaptogenesis following traumatic brain injury
Authors: Warren Kelly M , Lee Nancy N , Black Raiford T , Harris Lesley K , Phillips Linda L
Published: June 2021

Neuroglia play an important role in synaptogenesis after traumatic brain injury (TBI). The magnitude of glial response appears to be injury specific, potentially facilitating or attenuating synaptic recovery. Our prior studies also show that matrix metalloproteinases (MMPs) are differentially upregulated within reactive glia as a function of injury. Here, we contrasted glial response in models of adaptive (unilateral entorhinal cortex lesion; UEC) or maladaptive (combined fluid percussion injury and bilateral entorhinal cortex lesion; TBI+BEC) synaptic plasticity. Rats received UEC or TBI+BEC and were sacrificed 7d postinjury. Excised brains were processed for immunohistochemistry (IHC) with antibodies specific to astrocytes (GFAP), microglia (Iba‐1) and membrane‐bound MMPs, ADAM‐10 and MT5‐MMP. Confocal images revealed an increase in GFAP and Iba‐1 over deafferented neuropil in both models compared to uninjured controls. Microglial response was similar in the two models, however, TBI+BEC showed a greater GFAP increase across both dentate molecular and granule cell layers. Only GFAP+ cells were co‐labeled ADAM‐10 and MT5‐MMP. Results suggest that extent and distribution of TBI glial response is injury specific. Moreover, astrocytic ADAM‐10 and MT5‐MMP expression during reactive plasticity is a potential therapeutic target to facilitate synaptogenesis after TBI. Supported by: NIH grants NS44372, NS57758.
Estrogen worsens outcome from experimental stroke in aged rats
Authors: Leon Rachel L. , Li Xinlan , Rosen Charles L. , Huber Jason D.
Published: June 2021

Data from the Women's Health Initiative and other clinical trials have shown increased stroke severity in women taking hormone replacement therapy (HRT), despite evidence from animal studies demonstrating estrogen is neuroprotective. This dichotomy has spurred a debate about the safety and necessity of HRT. In the present study, we hypothesized that female rats supplemented with estrogen would suffer worsened outcome from experimental stroke due to altered post‐ischemic glycoprotein 130 (gp130) signaling. To test this hypothesis, female rats received chronic estrogen supplementation during aging and underwent occlusion of the middle cerebral artery with tissue plasminogen activator reperfusion at 18 months. Results demonstrate that aged female rats supplemented with estrogen suffer increased infarct size and higher mortality from ischemia/reperfusion. Estrogen supplementation also changed post‐ischemic mRNA levels of neuropoietic cytokines and gp130 signaling modulators in aged rats. Despite its anti‐inflammatory activity, estrogen appears detrimental to recovery from stroke in the aged brain. These findings suggest that chronic estrogen treatment play a role in worsened stroke outcomes in women taking HRT by altering post‐ischemic gp130 signaling pathways.
Increased prevalence of Anti‐Heparin Platelet factor 4 antibodies in patients with sepsis associated Disseminated Intravascular Coagulation
Authors: Thethi Indermohan , Al‐Duwaisan Laila , Cunanan Josephine , Khan Hussain , Hoppensteadt Debra , Kaul Inder , Fareed Jawed
Published: June 2021

The purpose of this investigation was to study the prevalence of anti‐ HPF4 antibodies in patients with sepsis associated DIC. A total of 727 baseline blood samples were obtained from patients recruited in the ART‐123 trial, which was designed to test the therapeutic efficacy of recombinant thrombomodulin in this indication. A sandwich based ELISA method (GTI, Waukesha, WI) was used to detect anti‐HPF4 antibodies. In this multicentric study, patient plasma was received at Loyola University Medical Center in frozen form and analyzed in batches. Of the 727 samples, 173 (23.8%) were found to be positive for the presence of anti‐HPF4 antibody (using the cut off criteria of OD>0.4, mean OD=0.653, SEM=0.032). 554 patients (76.2%) were found to be negative in accordance to the criterion (OD<0.4, mean OD=0.254, SEM=0.005). The absorbance ranges were then analyzed in four quartiles for the entire study cohort. There were 280 patients (38.5%) that fell in the first quartile (OD<0.25), 350 patients (48.1%) in the 2nd quartile (OD 0.25–0.50), 60 patients (8.2%) in the 3rd quartile and 37 patients (5.1%) in the 4th quartile. These observations clearly suggest that a significant number of these patients exhibit a high titer of the anti‐HPF4 antibody.
Histomorphometric characterization of thyroid follicles' areas on experimental hyperthyroidism and acupuncture treatment
Authors: Farinha Rodrigo , Cabral António , Machado Vanessa , Nolasco Varão , Cabrita A.M. Silvério
Published: June 2021

Hyperthyroidism is caused by excess synthesis and secretion of thyroid hormone by the thyroid, which increases the metabolic rate. The experimental model of hyperthyroidism is conducted by an administration of an high dose of levothyroxine for two weeks. The aim of this study is to contribute to the histomorphometric characterization of thyroid follicles' areas on experimental hyperthyroidism.

20 Wistar male rats were used, randomly distributed in four groups: control group, kept with no manipulation (Ctrl); group II, which received an administration of 250 mg/kg of levothyroxine by gavage 3 times a week (HT); group III received levothyroxine and acupuncture treatment (HT+AC); group IV, submitted only to the acupuncture treatment. After two weeks, all the animals were sacrificed and fragments from thyroid were collected and analyzed using ImageJ®. The points used for puncture were: ST09, ST36, HC4, RM23.

When compared with the control group, we observed more frequently large follicles on group IV than in control group. The animals of group II and III have more often smaller follicles than those observed in control group.

The results, although without statistical significance, suggest that acupuncture may have some effect on thyroid function.
Anticoagulant response variability in generic versions of argatroban. Dosing Implications
Authors: Thethi Indermohan , Hoppensteadt Debra , Cunanan Joesphine , Lewis Bruce , Fareed Jawed
Published: June 2021

To compare the anticoagulant responses, three generic versions of argatroban, argaron (A), Gartban (G) and slovastan (S) along with the branded product (BA) were assayed in various whole blood (ACT at a concentration of 2.5 ug/mL) and plasma clotting times (PT, APTT and TT at a concentration of 1ug/mL). In comparison to BA, the generic versions produced variable assay dependant responses in both the whole blood and plasma based assays. Moreover, the generic versions of argatroban also exhibited variations in the thrombin generation inhibition assays. When activated prothrombin complexes were used to neutralize the anticoagulant effects of argatroban, product based variations were evident. These differences were independent of the Ki value for the inhibition of purified thrombin. These results indicate that while in purified systems the potency of the generic and branded argatroban is comparable, in whole blood and plasma based systems, these drugs may differ which can be further complicated by pathologic status of patients.
Ethanol Interferes with Gene Regulatory Networks of Embryonic Stem Cells
Authors: Sanchez‐Alvarez Rosa , Gayen Saurabh , Vadigepalli Rajanikanth , Anni Helen
Published: June 2021

Gestational exposure to ethanol may promote abnormalities in the fetus, including growth retardation, facial dysmorphology and mental disability, under the umbrella of Fetal Alcohol Spectrum Disorders (FASD). We studied the interference of ethanol with normal embryonic development in embryonic stem (ES) cells. Mouse ES cells (E14Tg2A) were cultured in feeder‐free conditions, and converted to neural lineages in adherent monolayer in the presence or absence of ethanol. High throughput BioMark microfluidic 48.48 dynamic array chips (Fluidigm) measuring 2,304 real time quantitative PCR assays were used to assess levels of transcription factors and target genes involved in cell fate commitment during cell differentiation. Our data show that during ES cell differentiation i) genes regulating pluripotency and self‐renewal (Sox2, Nanog, Oct4, Klf4, Rex1, Dax1) were downregulated, iii) neuroectoderm and neural markers (e.g., Cxcl12, Nrip1) were upregulated, and ii) ethanol exposure delayed these changes. Network analysis points to nodes and edges of the gene regulatory networks perturbed by ethanol during early differentiation that may be responsible for aberrant neuroectodermal development in FASD.

Supported by TJU Pilot Research Award, Dept Funds, and Graham Foundation.
Spermatic Granuloma and Inflammatory Reactions in Epididymis of Adult Rats following Testosterone Depletion
Authors: Dutta Dibyendu , Park In , Guililat Hiwot , Hanson Laura , Sang Samuel , Mills Nathaniel
Published: June 2021

Depletion of testosterone (T), vasectomy or chemical insults are known to induce spermatic granuloma (SG) in epididymis, however, the exact mechanisms are unknown. We treated adult male rats with a Leydig cell specific toxicant ethylene dimethane sulfonate (EDS) to deplete T, causing SG formation in epididymis. Histological evaluation revealed the presence of testicular germ cells (TGC) in the caput of epididymis which were apoptotic. An increased proportion of tubular epithelial cells in caput were also apoptotic. Immunofluorescent staining showed extensive infiltration of monocytes/macrophages around the periphery of the shrunken tubules and SG. CD4+ and CD8+ lymphocytes were also detected inside the tubules and SG. In confirmation, quantitative real time PCR for mRNA of macrophage marker CD68, and lymphocyte markers CD4 and CD8 were increased in EDS treated epididymis compared to controls (19 fold, 3.5 fold and 7.5 fold respectively). Aquaporin 9 mRNA increased 11 fold in EDS treated epididymis which may be indicative of restricted testicular fluid flow into epididymis. These pathological conditions were reversed after T replacement in EDS treated rats. Thus, our data indicates that influx and accumulation of apoptotic TGCs in the epididymis due to the disintegration of the blood‐testis barrier and disruption of testicular fluid flow into epididymis may play a role in SG formation.
Reduced expression of tryptophan hydroxylase in the prefrontal cortex of a parkinsonian mouse model
Authors: Nayyar Tultul , Ferguson Marcus C , Ansah Twum A
Published: June 2021

OBJECTIVE

In 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated mouse model of Parkinson's disease (PD), we have shown a significant decrease in the density of beaded type serotonin (5‐HT) axons and concentrations of 5‐HT in the prefrontal cortex (PFC). Here we studied the protein level of tryptophan hydroxylase (TPH) and beta arrestin (βAR) 1 and 2 in the PFC in MPTP treated mice.

DESIGN AND METHODS

Male C57BL/6J mice were treated with 4 doses of MPTP 20 mg/kg (i.p.) at every 2 hours and sacrificed after 3 and 16 weeks. The protein expression for TPH, βAR‐1 and ‐2 in the PFC and TPH in the raphe was studied by Western blot. 5‐HT cell bodies in the raphe were studied by immunohistochemistry.

RESULTS

MPTP‐treated mice have shown a significant decrease in TPH protein level in PFC and a significant increase in the raphe nucleus. Protein expression for β‐AR1 and β‐AR2 were also significantly decreased in PFC. Immunohistochemistry of 5‐HT neurons in the raphe nuclei with TPH antibodies showed reduced intensity of staining for 5‐HT cell bodies in medial raphe (MR) and slightly increased in dorsal raphe (DR) after MPTP treatment. The reduced density of TPH‐labeled 5‐HT neurons in the MR correlated well with our earlier report of preferential decrease in the density of thick beaded 5‐HT axons in the PFC which originate from MR.

CONCLUSION

These results suggest that TPH is a major target of MPTP‐induced alterations of serotonergic function in PFC with a possible link to depression. Targeting TPH and related signaling events may afford therapeutic advantages for the management of depression in PD. Supported by NIH SNRP/NINDS grant NS041071.
Caveolin regulation of microglial activation and proliferation
Authors: Niesman Ingrid R , Patton Miranda , Zemke Nathan , Levy Karen , Ali Sameh S , Head Brian P , Patel Hemal H
Published: June 2021

Microglia are ramified cells serving as central nervous system (CNS) guardians. Following CNS injury or infection, microglia contract and cells adopts an amoeboid morphology capable of proliferation and migration. Though extracellular factors may be involved in this transformation, the control point for microglia activation or repression is unknown. Caveolin (Cav), a cholesterol binding protein, is expressed in inflammatory cells, yet the role of Cav in macrophage physiology is debatable. We propose that Cavs provide critical signaling regulatory points for injury‐induced microglial function. Global Cav‐1 knockout mice show increased hippocampal microglia compared to age matched littermates. Using a mouse microglia cell line (BV2) we investigated the role for Cavs in microglia function. We detected mRNA and protein for all three Cav isoforms (Cav‐1, ‐2, and ‐3) in BV2 cells. Cav‐1 protein was significantly reduced in inactive cells and localized to plasmalemmaand cytoplasmic vesicles (PM/CV). In activated cells, Cav‐1 appeared cytosolic or associated with PM/CV. In contrast, Cav‐3 was highly expressed in inactive cells and localized with processes and perinuclear. In the activated state, Cav‐3 expression decreased and localization was predominantly detected in PM/CV. The present findings suggest distinct regulation of microglial function is Cav isoform specific in CNS and Cav may be an effective inflammatory target in the setting of CNS injury
Gating of Pannexin 1‐Mediated ATP Release Channels by Mechanical Stress Stimuli
Authors: Tressler Andrea Michelle Boyd , Lai Charles , Naus Christian , Dubyak George
Published: June 2021

Most cells release ATP to extracellular compartments wherein ATP acts as a signaling molecule to regulate responses such as vasodilation and inflammatory activation. Pannexin‐1 (Panx1) hemichannels have been proposed as major conduits for ATP efflux in response to proapoptotic stimulation or mechanical deformation of the plasma membrane. Recent studies have demonstrated that caspase‐mediated cleavage of the Panx1 C‐terminus is a novel gating mechanism for this channel in apoptotic cells. In this study we examine the possible role of caspases or other proteases in the acute ATP release induced by non‐apoptotic mechanical stress stimuli in rat C6 glioma cells (which lack native Panx1) versus C6 cells stably transfected with Panx1 (C6‐Panx1). C6 cells were mechanically stimulated by medium exchange or by thrombin (which triggers rapid reorganization of the actin cytoskeleton) to elicit acute ATP release. Only minor ATP release was observed in control C6 cells in response to either medium exchange or thrombin addition. In contrast, medium exchange triggered robust ATP release from C6‐Panx1 cells. Current experiments are testing whether thrombin triggers similar ATP release in C6‐Panx1 cells and whether proteolysis of Panx1 mediates gating of these ATP release channels in response to mechanical stimulation.
G protein‐coupled receptor (GPCR) regulation of cardiac fibrosis
Authors: Snead Aaron Nathan , Insel Paul A
Published: June 2021

Cardiac fibrosis occurs in advanced age, after acute injury (e.g. myocardial infarction) and in disease states (e.g. diabetes mellitus). Fibrosis is characterized by the transformation of fibroblasts to myofibroblasts, which contribute to the excessive accumulation of collagen‐rich extracellular matrix and can result in reduced cardiac function. To identify novel therapeutic targets for cardiac fibrosis we used an unbiased approach (GPCR RT‐PCR array) to define the most abundantly expressed GPCRs that may regulate the fibroblast/myofibroblast transformation in rat cardiac fibroblasts (rCFs). Previous studies have shown that analogs of the second messenger cAMP can reverse or inhibit the phenotype induced by the pro‐fibrotic agents TGF‐β1 and angiotensin II. We identified protease‐activated receptor 1 (PAR1) as the most abundant GPCR in rCFs and found that activation of PAR1 elevates the expression of a variety of pro‐fibrotic markers via at least two distinct post‐receptor mechanisms. Additionally, activation of abundant Gs‐linked GPCRs, whose activation raises intracellular cAMP levels, identifies new potential targets and mechanisms by which cAMP inhibits rCF transformation. These data, which define the level of expression of GPCRs in rCFs and mechanisms for their actions provide a proof‐of‐principle that such an approach can identify novel disease‐relevant GPCR targets. Supported by NIH.
Phospholipase Cε Regulates Multiple Agonists‐Induced Cardiomyocyte Hypertrophy in Neonatal Rat Ventricular Myocytes By Binding To mAKAP (Muscle A‐Kinase Anchoring Protein) And Generating Local IP3‐Dependent Nuclear Calcium Release
Authors: Zhang Lianghui , Malik Sundeep , Park Keigan M , Kapiloff Michael S , Yule David I , Smrcka Alan V
Published: June 2021

Phospholipase Cε (PLCε) is a bifunctional PLC isoform that contains a PLC catalytic, Ras association (RA), and guanine nucleotide exchange factor (GEF) domains. To determine if PLCε plays a direct role in regulating cardiomyocyte hypertrophy, neonatal rat ventricular myocytes (NRVMs) were infected with adenoviruses expressing PLCε siRNA. PLCε siRNA infection of NRVMs inhibits Endothelin‐1 (ET‐1), isoproterenol (ISO) and insulin like growth factor‐1 (IGF‐1) induced protein synthesis and ANF (Atrial natriuretic peptide) mRNA expression – indexes of cardiac hypertrophy. These data indicate that PLCε signaling downstream of ET‐1R, βAR and IGFR is important for cardiac hypertrophy. PLCε bound to mAKAP (muscle A‐kinase anchoring protein), which localizes bound proteins to the nuclear envelope of myocytes. Overexpression of PLCε‐RA1 domain disrupts PLCε binding to mAKAP. Overexpression of the PLCε‐RA1 domain in NRVMs significantly reduces ET‐1, ISO and IGF‐1‐induced hypertrophy. To examine the role of PLCε and PLCε‐mAKAP scaffolding at the nuclear envelope, we measure nuclear Ca2+ release. PLCε siRNA and PLCε‐RA1 domain overexpression reduce ET‐1‐induced nuclear Ca2+ release in NRVMs. These results suggest that PLCε, scaffolded at the nuclear envelope, responds to multiple upstream signals to regulate cardiomyocyte hypertrophy via IP3‐ dependent nuclear Ca2+ release.