bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide expression and purification to elucidate the mechanism of vascular α‐2C adrenoceptor translocation
Authors: Fidelibus Robert , Jeyaraj Selvi C , Unger Nicholas T , Biesiadecki Brandon J , Chotani Maqsood A
Published: June 2021

In response to physiological cold temperatures cutaneous blood vessels constrict to reduce heat loss. This constriction is mediated by smooth muscle G protein‐coupled α2C‐adrenoceptors (α2C‐ARs) which translocate from the transGolgi to the plasma membrane upon cooling. We hypothesize that this translocation is due to protein‐protein interaction between α2C‐AR C‐terminus and the protein filamin‐2. The goal of this study is to test this mechanism of receptor translocation experimentally by decoy peptide interference. We predict that interfering with this interaction will inhibit translocation of α2C‐ARs and subsequently receptor function.

The DNA sequence encoding 11 amino acids of the cell‐permeable HIV transactivator of transduction (TAT) domain was fused in frame with the α2C‐AR C‐terminus to generate the p‐TAT‐α2C‐AR‐ C‐terminus bacterial expression vector. BL21‐CodonPlus competent cells were used to optimize peptide expression at 30°C. The parameters tested were bacterial growth, presence of antibiotics, induction time, cell lysis buffers, sonication and salt precipitation conditions. Peptide expression was confirmed by Western blot and Coomassie techniques. The peptide will be purified by ion‐exchange chromatography, fluorescently labeled for tracking and delivered to human cutaneous smooth muscle cells to assess cellular activity for endogenous α2C‐ARs.
Endothelial biomarkers as indicators of cardiovascular complications in young patients with arterial hypertension
Authors: Lyamina Nadezhda P. , Lyamina Svetlana V. , Downey H. Fred , Manukhina Eugenia B.
Published: June 2021

This study evaluated nitric oxide (NO) and endothelin‐1 (ET‐1) as clinically relevant biomarkers in young patients with early arterial hypertension (AH). Male patients (n=65, 20–45 years of age) with AH duration <6 years and blood pressure (BP) <160/100 mm Hg were studied. A control group consisted of 32 age‐matched healthy subjects. Endothelium‐dependent vasodilation (EDVD) was evaluated by measuring post‐occlusion reactive hyperemia in the brachial artery. NO production was evaluated by plasma concentration and 24‐hour urinary excretion of nitrite + nitrate. Plasma concentration of ET‐1 was measured by immunoenzyme assay. The plasma NO/ET‐1 ratio was calculated. EDVD was 12.3% lower in patients with AH than in healthy subjects, and this decrease correlated with the duration of AH. The NO/ET‐1 ratio was significantly lower in the AH group (2.0±0.4 vs 6.2±0.6, p<0.001), and the NO/ET‐1 ratio decreased with AH duration. NO/ET‐1 values below 3.0 were associated with atypical diurnal BP profile and cardiac arrhythmias. NO/ET‐1 values below 1.0 were associated with severe cardiovascular complications. Thus, the ratio of NO to ET‐1 appears to be a useful marker for cardiovascular complications in young patients with arterial hypertension.
Alterations of T cell receptor Vβ chain usage in angiotensin IIinduced hypertension
Authors: Trott Daniel , Chen Wei , Harrison David
Published: June 2021

Stimulation of the T cell receptor (TCR) with a specific antigen leads to clonal expansion of cells with identical TCRs. In contrast, if multiple antigens are presented, the T cells with multiple TCRs proliferate. We have previously shown that T cells play a critical role in the genesis of hypertension, and that activated T cells infiltrate the vasculature and the kidney to promote vasoconstriction and sodium retention. We sought to determine if hypertension promotes a mono‐ or polyclonal T cell response. We produced hypertension in C57Bl/6 mice by chronic (2 week, 490 ng/kg/min) infusion of angiotensin II and used vehicle infused mice as controls. Nested PCR was employed to identify TCR subfamilies of the Vβ chain in the kidney and vasculature. Only 4 of the 24 Vβ chain subfamilies were present in >50% of kidneys from controls, whereas 12 of the 24 were present in >50% of the kidneys from angiotensin II infused mice. Notably, Vβ4 was present in 2 of 8 sham and 6 of 8 Ang II samples and Vβ17 was present in 1 of 8 sham and 5 of 8 Ang II samples. In mesenteric vessels, 18 of 24 Vβ subfamilies were present in >50% of samples from controls and 15 of 24 were present in >50% of samples from Ang II samples. These results suggest that hypertension results in polyclonal T cell expansion in the kidney, likely due to the generation of multiple neoantigens.
Structural Biology of Methylation‐mediated Epigenetic Regulation
Authors: Patel Dinshaw J.
Published: June 2021

The lecture will present our research on recognition events controlling epigenetic regulation mediated by methylation marks on histone tails and at CpG sites on DNA.

Specific topics will include recognition of methyllyisne marks by reader elements in ADDATRX (Haitao Li), TRIM24/33 (Zhanxin Wang) and MLL1 (Zhanxin Wang and Jikui Song), erasure of lysine methylation marks by the KMD2A jumonji family lysine demethylases (Zhongjun Cheng), and DNMT1‐mediated active and autoinhibitory mechanisms of maintenance DNA methylation (Jikui Song).

Collaborators: Yang Shi (Harvard) and Haitao Li (Tsinghua) on ADDATRX, C. David Allis (Rockefeller) on MLL1, Michelle Barton (M. D. Anderson) on TRIM24, Joan Massague (Sloan‐Kettering) on TRIM33, and Or Gozani (UCSF) on KMD2A.
Electrogenicity and Anion Selectivity of the Sodium/Bicarbonate Transporter NBCe1 Determined by a Single Mechanism
Authors: LEE SOOJUNG , CHOI INYEONG
Published: June 2021

Electrogenic transporters move net charge across the membrane and generate currents. The mechanism of net charge movement involves interactions between ions and the transporter in the transmembrane electric field. We previously reported that an aspartate (Asp555) in transmembrane domain (TM) 5 of NBCe1 serves as an anion selectivity site. This finding is surprising because a glutamate is present at the corresponding site of all electroneutral sodium/bicarbonate transporters and thus Asp555 is expected to be linked to electrogenicity. Here, we tested whether this Asp/Glu replacement is responsible for both ion selectivity and electrogenicity of Na/HCO3 transporters. We changed local charges near Asp/Glu555 and determined their effects on HCO3− and Cl− currents by two‐electrode voltage clamp. Replacing Asp555 of NBCe1 with a Glu produced a Cl− current in addition to a HCO3− current. In contrast, replacing entire TM 5 of NBCe1 with Glu‐containing TM 5 of the electroneutral transporter NBCn1 produced only a small HCO3− current. The current‐voltage plot revealed outward HCO3− currents at all voltages and a small positive conductance. The Cl− current was rescued when a positively charged Lys was introduced at position 558 (located on the same face of helical TM5) in this chimeric transporter. Thus, anion selectivity appears to be determined by a charge interaction between Glu555 and Lys558. These results indicate that the amino acid compositions at the two sites determine the translocation of HCO3−, Cl− or none, which leads to the transporter's electrogenicity or electroneutrality. Our data provide novel evidence for a single mechanism determining both electrogenicity and ion selectivity of Na/HCO3 transporters.
Cardiac myocyte‐specific deletion of Heat shock protein 10 results in mitochondrial dysfunction and mortality
Authors: Wang Hong , Ouyang Kunfu , Kim Yun-Kyung , Scott Brian Thomas , Banerjee Indroneal , Mu Yongxin , Chen Ju , Dillmann Wolfgang H. , Suarez Jorge
Published: June 2021

Heat shock proteins (HSPs) are molecular chaperones that prevent protein damage and proteolysis. HSP10 forms mitochondrial (Mito) chaperoning complexes with HSP60 to maintain protein quality control. Studies in vitro suggest HSP10 plays a role in normal Mito function; however, its physiological function in the heart in vivo remains to be determined. To investigate the function of cardiac HSP10, we generated a floxed allele of the Hspe1 gene, and found that cardiac specific, tamoxifen‐inducible deletion of HSP10 in adult mice resulted in death between 12 and 18 weeks after HSP10 deletion. Increased mortality after HSP10 deletion was associated with Mito dysfunction. For instance, function of mitochondrial respiratory chain complex I was decreased by 26% as early as 6 weeks post tamoxifen treatment. In addition, there was a dramatic reduction in levels of Mito respiratory complex subunits including CI‐NDUFA9, CII‐30, COX I, COX IV, and COX Vb; although no change was observed in HSP60 at the same stage. Our results suggest mitochondrial damage may be the primary pathological cause of the death. In conclusion, HSP10 plays a vital role in regulating mitochondrial protein homeostasis.
Modulation of baroreceptor afferent terminal excitability and reflex responses by phospholipase D‐coupled metabotropic glutamate receptors
Authors: Paton Julian Francis , Banks Robert W , Bewick Guy S
Published: June 2021

We have demonstrated glutamate released from clear synaptic‐like vesicles (SLVs) modulates mechanoreceptor excitability in Ia primary afferent endings of rat muscle spindles acting on phospholipase D‐linked metabotropic glutamate receptors (PLD‐mGluRs; Bewick et al. 2005 J. Physiol 562, 381‐). We have tested whether the SLVs in baroreceptor endings (Krauhs, 1979 J. Neurocytol. 8:401‐) show a similar mechanism that modulates both afferent discharge and reflex‐evoked responses. Baroreceptors were stimulated by ramp increases in arterial pressure while either afferent discharge or reflex changes in heart rate and sympathetic nerve activity were measured after superfusion of the aortic arch with either the PLD‐mGluR antagonist PCCG‐13 (20μM) or glutamate (10mM) in situ. PCCG‐13 decreased baroreceptor afferent activity by 35% (P<0.05) and glutamate increased discharge by 42% (P<0.05) relative to control levels. Cardiac baroreceptor reflex gain was was attenuated reversibly by PCCG‐13 by 51% from 2.8+0.4 bpm/mmHg (P<0.05). Baroreceptor reflex‐mediated sympathoinhibition was also depressed. Glutamate applied to the aortic arch mimicked a baroreflex response. These data indicate autogenic glutamate release from baroreceptor terminals regulate afferent discharge through PLD‐mGluRs to modulate reflex cardiac and sympathomotor responses. MRC funded research.
Altered brainstem neuromodulators relevant to central chemoreceptor function in Brown Norway (BN) rats
Authors: Hodges Matthew Robert , Mouradian Gary , Miller Justin , Muere Clarissa , Forster Hubert Vincent
Published: June 2021

Brown Norway (BN) rats exhibit a selective deficit in the hypercapnic ventilatory response (HVR) compared to Dahl Salt‐sensitive (SS) rats, and show differential expression of various pH‐sensitive channels in postulated sites of central CO2/H+chemoreception, including the serotonergic medullary raphé and nucleus of the solitary tract (NTS). Here, we test the hypothesis that brainstem neuromodulators, such as serotonin (5‐HT), dopamine (DA), and norepinephrine (NE), and their metabolites, are altered in BN rats compared to SS rats. To test this hypothesis, we rapidly removed whole brains from adult (>7 weeks of age) BN (n=6) and SS (n=6) rats, and separated it into 5 major brain regions: 1) medulla, 2) pons/midbrain, 3) cerebellum, 4) hypothalamus and 5) forebrain for high‐performance liquid chromatography (HPLC) analysis. In separate animals, we transcardially‐perfused and fixed brains for cryostat sectioning and immunostaining for 5‐HT (TPH‐expressing) or TH‐expressing neurons. We found that tissue levels of 5‐HT were significantly (p<0.05) lower in BN medulla, cerebellum and hypothalamus, and 5‐HIAA levels were lower (p<0.05) in medulla, pons/midbrain, hypothalamus and forebrain compared to SS rat tissues. In addition, tissue NE levels were also lower (p<0.05) in the hypothalamus of BN rats, but not different in any other region compared to SS rats, and we found no differences in DA in all tissues studied. Despite the reduced 5‐HT, 5‐HIAA, and NE levels, we did not find a difference in counts of TPH+ or TH+ neurons throughout the medulla. These data suggest deficiencies in 5‐HT and NE may be related to altered neuronal function rather than altered cell numbers in BN rats, which may contribute to the deficit in the HVR in BN rats. Supported by NIH HL097033 and Veterans Affairs.
Pre‐Botzinger Complex (PBC) Plays An Important Role in the Hyperventilatory Response to Intravenous Injection of 5HT1AR Agonist in Anesthetized Rats
Authors: Zhuang Jianguo , Zhang Cancan , Xu Fadi
Published: June 2021

Systemic administration of 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (DPAT), a 5HT1AR agonist, causes a hyperventilation via elevating fR in animals. It also prevents peripheral and central mu‐opioid receptor‐induced apnea (Zhuang et al, EB 2010; Sahibzada et al, JPET 2000). Because the PBC containing 5HT1ARs is critical in respiratory rhythm, we examined whether local 5HT1ARs are involved in the evoked hyperventilation. In anesthetized rats, the cardiorespiratory responses to systematic DPAT challenge (100 μg/kg, iv) were performed 1 hr before and 5 min after bilateral microinjection of WAY‐100635, a selective 5HT1AR antagonist (20 mM, 10 nl), into the PBC via a ventral approach. Our results showed that: 1) DPAT doubled VE primarily via elevating fR associated with a hypotension (−34%) and little change in HR, which recovered ~60 min later; 2) microinjection of WAY‐100635 into the PBC did not alter baseline cardiorespiratory variables, but strikingly attenuated (50% reduction) the DPAT‐induced hyperventilation and shortened the recovery period to ~30 min with the hypotension unchanged; and 3) the same microinjection made 1 mm rostral to the PBC or vehicle microinjected into the PBC failed to affect this evoked hyperventilation. Our data suggest that systemic DPAT challenge causes the hyperventilation largely through acting on PBC 5HT1ARs (Supported by HL 107462 and ALA RG‐191095‐N).
Up‐regulation of Complex I S‐glutathionylation by eNOS knockout Is Mediated by Increasing Oxidant Stress and Protein Thiyl Radical in Mitochondria
Authors: Chen Yeong-Renn , Kang Patrick T. , Chen Chwen-Lih
Published: June 2021

In response to oxidant stress, mitochondrial complex I was reversibly S‐glutathionylated. To gain deep insights into the mechanism, complex I S‐glutathionylation (PrSSG) was induced by O2•− in the presence of GSH. Immuno‐spin trapping and MS indicated that formation of complex I protein thiyl radical intermediate (PrS•) mediates PrSSG. In myocytes treated with menadione to trigger mitochondrial O2•− production, complex I PrSSG and PrS• were increased, supporting the hypothesis of oxidant stress induced reactive protein thiyl radical to mediate complex I PrSSG. The hypothesis was further tested in the animal model of eNOS(−/−). The mitochondria isolated from eNOS(−/−) mouse heart showed a marked dysfunction with impaired state 3 respiration, declining respiratory index, and deceasing enzymatic activities of ETC. Further biochemical analysis and EPR measurement indicated a defective aconitase activity, increasing glutathione peroxidase activity, and a marked increase in the O2•− generation activity. These results suggested increasing ROS production and oxidant stress in the mitochondria of eNOS(−/−) mouse heart. When the complex I was immune‐precipitated from the heart and probed with anti‐GSH antibody, PrSSG was significantly enhanced, thus supporting mediation of complex I PrSSG by reactive protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function. (Supported by NIH grant HL083237)
FosB in NTS contributes to persistent increase in mean arterial pressure during exposure to chronic intermittent hypoxia
Authors: Wu Qiong , Cherry Brandon , Mifflin Steve
Published: June 2021

FosB is a member of the transcription factor Activator Protein C1 (AP‐1) family, has low constitutive expression in the CNS and is induced following chronic stimulation such as chronic intermittent hypoxia (CIH). FosB heterodimerizes with JunD to regulate gene expression at AP‐1 sites. We hypothesize that FosB in NTS contributes to the persistent increase in mean arterial pressure (MAP) during exposures to CIH. We injected a GFP labeled, AAV‐mediated ΔFosB dominant‐negative antagonist ΔJunD into three sites of the NTS of 3 Male Sprague Dawley rats, 100nl per site. Six rats were injected with AAV‐GFP as sham controls. Following injections, rats were exposed to CIH for 8h/day, for 7 days. MAP and HR were recorded using telemetry. The dominantnegative ΔJunD did not alter MAP or HR responses during the daytime exposure to CIH. During the dark period, in the absence of hypoxia, MAP was increased in the sham group from 102 ± 2 mmHg to 108 ± 1 mmHg following the 7 day exposure to CIH. In the group receiving the dominant‐negative ΔJunD, MAP measured during the dark increased from 102 ± 1 mmHg to 104 ± 2 mmHg following the 7 day exposure to CIH. These data indicate that the transcription factor FosB within the NTS contributes to the persistent increase in MAP induced by exposures to CIH.
Evidence that glial cells modulate synaptic transmission in the NTS neurons sending projections to RVLM
Authors: Accorsi-Mendonça Daniela , Bonagamba Leni GH , Machado Benedito H
Published: June 2021

The peripheral chemoreflex activation produces autonomic and respiratory responses. In previous study we verified that ATP modulates the glutamatergic transmission onto NTS neurons sending projections to RVLM, which are related to the sympathoexcitatory component of chemoreflex. Here, we evaluated the role of glial cells in ATP release and the purinergic subtype receptors involved with this modulation. Adult rats received fluorescent tracer microinjections into RVLM and 7 days later the labeled NTS neurons sending projections to RVLM were identified in brainstem slices. TS evoked excitatory pos‐synaptic currents (TS‐eEPSCs) were recorded using patch clamp. Inhibition of glial cells with fluoracetate (FAC− 1mM) decreased the amplitude of TS‐eEPSCs and this effect was independent of glutamine in the bath (FAC: − 56 ± 0.06%, n=6; FAC + glutamine: −65 ± 0.11%, n=10). iso‐PPADS (100 μM), a P2X receptor antagonist, or A317491 (10 μM), a P2X3 or P2X2/3 receptor antagonist, produced similar decrease in the amplitude of TS‐eEPSCs (iso‐PPADS: −58 ± 7%, n=6; A317491: −57± 5%, n=5%). The subsequent addition of iso‐PPADS in the bath during FAC perfusion did not alter the glial inhibition effect on TS‐eEPSCs (FAC: −58 ± 11%; iso‐PPADS + FAC: −45 ± 6%, n=5). We conclude that glial cells modulate the synaptic transmission in the NTS neurons sending projections to RVLM. Supported by FAPESP and CNPq.
Electrospun Proteoglycan Matrices for Tracheal Tissue Engineering
Authors: Hinderer Svenja , Bayrak Alexandra , Schesny Marianne , Hampel Martina , Seifert Martina , Schenke-Layland Katja
Published: June 2021

Decorin (DCN) is a proteoglycan (PG) present in the extracellular matrix (ECM) of many tissues, including the trachea. Electrospinning is a suitable method for three‐dimensional (3D) scaffold fabrication to mimic the ECM's fibrous structure. In this study, we generated electrospun DCN‐polymer hybrids to obtain scaffolds that allow human tracheal cell (hTC) seeding.

Methods

DCN containing scaffolds were fabricated and characterized using scanning electron microscopy, contact angle measurements and dynamic mechanical analyses. Immunofluorescence (IF) staining was performed to detect DCN within the scaffold. For cell‐matrix interaction studies, we seeded primary hTCs and confirmed their phenotype by using immunocytochemistry.

Results

Analyses of the 3D substrates revealed a fibrous scaffold with a random fiber orientation. An average fiber diameter of 0.42 ± 0.20 μm and a pore size of 14.4 ± 6.4 μm2 was determined. Antibody staining for DCN showed the PG randomly distributed throughout the entire scaffold. After cell seeding, IF staining with an antibody against FOXJ1 revealed that ciliated hTCs adhered and proliferated on the scaffolds.

Conclusion

Here, we showed the successful electrospinning of PGs in combination with polymers to generate a fibrous scaffold with features of the natural ECM. Our data suggest that these substrates are excellent for applications in regenerative medicine.

Grant Funding Source: Fraunhofer‐Gesellschaft (Attract 692263 to K.S.‐L)
A microfluidic bioreactor for epithelial cell studies under fluid shear stress
Authors: Ferrell Nicholas , Ricci Kevin B. , Desai Ravi R. , Groszek Joseph , Marmerstein Joseph T. , Fissell William H.
Published: June 2021

We have developed a multilayer microfluidic bioreactor to evaluate polarized epithelial cell behavior under controlled fluid shear stress. The system consists of two microfluidic chambers separated by a synthetic porous membrane which provides a substrate for cell grown and allows apical to basolateral solute transport. The system has been used to evaluate a number of shear stress‐dependent cellular behaviors in kidney epithelial cells. We found that primary human renal epithelial cells undergo cytoskeletal rearrangement when exposed to shear stress. Endocytosis, metabolism and secretion of albumin were also evaluated using the bioreactor. Fluid shear stress increased the cellular capacity for apical uptake of albumin in immortalized proximal tubule epithelial cells. Following internalization, albumin was metabolized into small molecular weight fragments and excreted into the apical and basolateral compartments. This microfluidic bioreactor is a useful tool for evaluating kidney epithelial cell behavior in response to fluid shear stress and may have broader applications for studying a variety of shear stress sensitive cell types. This work was supported by the Wildwood Foundation and a T32 training grant from the National Institutes of Health (T32‐DK‐007470–25).
Caffeic Acid Phenethyl Ester protects burn wounds against oxidative stress and improves wound healing
Authors: Monte-Alto-Costa Andrea , Santos Jeanine Salles
Published: June 2021

Caffeic acid phenethyl ester (CAPE) is an active component of propolis, and has been shown to have anti‐inflammatory and antioxidant effects; in this study we studied its effects on burn wound healing. Twenty one female Wistar rats were divided in three groups: control (no burn)+CAPE, burn and burn+CAPE. A third‐degree burn was created. The CAPE treatment (10 μmol kg‐1; i.p.) began immediately after burn and lasted until euthanasia (14 days after burn). Fourteen days after wounding the burn+CAPE group presented greater wound contraction when compared to burn group. In plasma, lipid peroxidation was higher in burn group compared to control and burn+CAPE group. In wounds, burn+CAPE group presented a smaller lipid peroxidation than burn group. Carbonyl levels in plasma and lesions of the burn group were increased when compared to control group. In burn+CAPE group carbonyls levels were greater than the control only in wounds. Catalase activity was also analysed, and burn+CAPE group presented greater activity when compared to burn, but without difference when compared to the control group. It was also observed that both burn and burn+CAPE groups present a higher myeloperoxidase activity than control group, but the burn+CAPE group presented smaller myeloperoxidase activity compared to burn group. CAPE treatment improved wound contraction and reduced the oxidative damage of burns.

Support: CAPES
FOXC1 regulates BMP‐SMAD activity in a context dependent manner during osteogenic development events
Authors: Berry Fred B , Mirzayans Freda
Published: June 2021

The forkhead box transcription factor FOXC1 is necessary for bone morphogenetic protein (BMP)‐mediated induction of early skeletogenic patterning events. We hypothesize that FOXC1 and SMAD proteins interact to regulate BMP signalling necessary for normal skeletal development. First we demonstrate that FOXC1 and SMAD5 physically interact through co‐immunoprecipitation experiments. Next we show that BMP2 enhances activation of a FOXC1‐responsive reporter gene. We tested whether FOXC1 could mediate signalling of a BMP2‐responsive reporter (2xBRE‐luc). We find that on its own, FOXC1 has no affect on activation of 2xBRE‐luc. Surprisingly, when BMP2 is added, FOXC1 inhibits activation of this reporter. Moreover, FOXC1 prevents activation of this BMP‐responsive element when SMAD4 and SMAD5 are co‐transfected along with FOXC1. To test whether FOXC1 impacted BMP2 mediated osteogenic differentation, we created C2C12 cell lines stably expressing FOXC1. We find that cells expressing FOXC1 differentiated into alkaline phosphatase‐positive osteoblasts in the absence BMP2, yet treatment of FOXC1 expressing cells with BMP2 led to no appreciable increase in alkaline phosphatase activity. Finally we show that FOXC1 prevents activation of Runx2 expression by BMP2. Together these data indicate that FOXC1 and SMAD5 proteins interact to regulate BMP‐signalling in a context dependent manner.
Epigenetic Regulation in Leukemia Cell Differentiation
Authors: Zhang Kangling , Filippova Maria , Filippov Valery , Duerksen-Hughes Penelope J.
Published: June 2021

Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics‐based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol‐12‐myristate 13‐acetate‐induced differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin‐coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were upregulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down‐regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down‐regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase II‐associated FACT‐RTF1‐ PAF1 complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation.
Sequence Selective Recognition of Double Helical RNA Using Nucleobase‐Modified PNA
Authors: Rozners Eriks , Zengeya Thomas , Muse Toyosi , Gupta Pankaj
Published: June 2021

The important role that non‐coding RNA plays in cell biology makes it an attractive target for molecular recognition. The surface of an RNA helix is relatively uniform, dominated by negatively charged phosphates and presents little opportunity for the traditional shape selective molecular recognition. We envisioned that a hydrogen bond mediated major groove triple helix formation could provide the most straightforward sequence selective recognition of double helical RNA. Using isothermal titration calorimetry we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (Ka > 107) and sequence selectively to polypurine tract of double helical RNA. The data suggest that the binding mode is a sequence selective triple helix formation. Although RNA does not have long polypurine tracts, it is common to find stretches of seven and more purines interrupted by one or two pyrimidines in microRNAs and ribosomal RNAs. We found that such pyrimidine interruptions in the polypurine tracts of biologically relevant RNAs can be recognized in a sequence specific manner using nucleobase‐modified PNA under physiologically relevant conditions. Our results have implications for development of biochemical probes to study function of non‐coding RNAs and design of novel therapeutic approaches based on sequence selective inhibition of microRNAs. Supported by NIH R01 GM071461
Cisplatin‐induced, coordinated transcriptional reprogramming of neurotrypsin and other proteases in cisplatin‐resistant ovarian cancer cells
Authors: Garcia-Bassets Ivan , Almenar-Queralt Angels
Published: June 2021

The contribution of extracellular proteolysis to tumor growth, invasion, and metastasis has been recognized for many years. Deregulation of this process remodels normal tissue microenvironment, facilitating malignant transformation, cell migration and invasion, and sometimes resistance to chemotherapy. The mechanisms involved in these events have been extensively studied, but generally focused on individual proteases. We will show that cisplatin exposure, which is a common chemotherapy strategy, induces rapid and coordinated transcriptional reprogramming of 13 genes encoding proteolytic activities in cisplatin‐resistant ovarian cancer cells. These genes include 4 serine proteases, 4 kallikrein, and 5 matrix metallopepsidases. We will cover the transcriptional events involved in this deregulation, including the role of specific epigenetic modifiers at the promoters of these genes. We will also show cellular effects resulting from the suppression of these events, with emphasis on the activity of the serine protease neurotrypsin and its known substrate, the heparan sulfate proteoglycan agrin. Neurotrypsin cleaves agrin generating three fragments: N‐terminal agrin, agrin‐90, and agrin‐22. Together, we propose that cisplatin exposure induces coordinated deregulation of extracellular proteases contributing to escape drug toxicity in cisplatin‐resistant ovarian cancer cells.
Impaired lysosomal maturation of pro‐cathepsin D to active cathepsin D in a childhood neurodegenerative lysosomal storage disease
Authors: Chandra Goutam , Saha Arjun , Moralle Matthew R , Zhang Zhongjian , Sarkar Chinmoy , Peng Shiyong , Mukherjee Anil B
Published: June 2021

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of childhood neurodegenerative lysosomal storage disorders. The infantile NCL (INCL), the most lethal among all NCLs, is caused by inactivating mutations in the palmitoyl‐protein thioesterase‐1 (PPT1) gene. PPT1 is a lysosomal enzyme that catalyzes the cleavage of thioester linkage in palmitoylated (S‐acylated) proteins facilitating their degradation. Although a suggested mechanism of INCL pathogenesis is that PPT1‐deficiency causes abnormal accumulation of S‐acylated proteins in lysosomes, the precise molecular mechanism remains poorly understood. We report here that oxidative‐stress mediates overexpression of lysosomal procathepsin D (pro‐CTSD) via increased production of an oxidative stress‐sensitive transcription factor, CEBP‐∂. Interestingly, while pro‐CTSD‐mRNA and protein expression is elevated in the brain of Ppt1‐knockout mice, the maturation and activity of lysosomal CTSD were impaired. Our results suggest that due to the increase in lysosomal pH in PPT1‐deficient cells the activity of cathepsin L, which is required for cathepsin D processing, is disrupted and consequently, both the levels of mature cathepsin D and its activity are impaired. We propose that this abnormality impairs the degradation of lysosomal cargo, which contributes to INCL pathogenesis. This work was supported by Intramural Research Program of NICHD.
Fluorescence detection of obligatory conformations in catalysis and control of NOS
Authors: Salerno John C , Baquerizo Jose L , Harris Kyle D , Hopper Benjamin L. , Enweani Chioma Grace , Umejiego Ezigbobiara Nkasiobi , Razdan Raj , McMurry Jonathan L. , Chrestensen Carol A , Ghosh Dipak K
Published: June 2021

Nitric Oxide synthases produce NO as a signal and as a cytotoxin in immune response. In the tethered shuttle model for NOS reductase kinetics, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic sites. FMN fluorescence is sensitive to NOS conformation; eNOS and nNOS holoenzymes have states with lifetimes of ~4.3ns, 1 ns, and 90 ps. Two domain oxyFMN constructs (truncated before the FAD binding domain) and holoenzyme share open conformations with lifetimes of ~4.3 ns. The holoenzyme majority state has a lifetime of ~90 ps due to FAD/FMN proximity as in crystal structures (Wang et al, Garcin et al). In oxyFMN ~25% of FMN has a lifetime of 0.9 ns, which we attribute to quenching by heme. Calmodulin activation of NOS releases the FMN binding domain from the ‘dehydrogenase’ complex, increasing the populations of the long lifetime states. Calmodulin binding shortens the average lifetime of oxyFMN constructs, because CaM promotes association of the FMN binding domain with the oxygenase domain, and favors the 1 ns state over the 4.3 ns state. Similar effects help explain modulation of NOS activity by covalent modification and protein‐protein interactions, e.g., in the HSP90‐eNOS complex.
Elucidation of mechanisms of biochemical regulation of fumarase activity under physiological conditions
Authors: Mescam Muriel , Vinnakota Kalyan C , Beard Daniel A
Published: June 2021

The enzyme fumarase catalyses the reversible mechanism of hydration/dehydration of fumarate to malate. This reaction is a critical step of the tricarboxylic acid (TCA) cycle and hence of the synthesis of ATP in metabolism. In a recent study, we have identified a catalytic mechanism for the enzyme in various conditions of buffer concentrations, pH and initial substrate concentrations [1]. In the current study, we investigate the influence on the kinetics of fumarase, of various anions such as phosphate, citrate, isocitrate, ATP and ADP. An eleven‐state computational model is fitted to time‐courses of fumarate hydration, obtained under physiological conditions in terms of ionic strength, pH, temperature and concentrations, in order to determine the potential inhibitory/activating effect of each anion on the kinetics of fumarase. This combination of experiments and computational modeling provides a quantitative understanding of the enzyme's catalytic mechanism.

This work is supported by the NIH Grant HL094317.
Understanding the Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN)
Authors: Crawford Patrick , Kimberley Butnik , Kassl Christopher , Nelson Nicole , Anliker Mitchell , Golich Frank C , Crowder Michael W
Published: June 2021

Aminopeptidase N (PepN) from E. coli is an M1 class, Zn(II)‐dependent peptidase with broad specificity. PepN was mechanistically characterized in an effort to understand the role of tyrosine 381. pH Dependence kinetic studies revealed a log kcat versus pH and kcat/KM versus pH curves with inflection points at a pH of 9.0. Solvent isotope studies suggest at least one rate‐limiting proton transfer in the mechanism. In an effort to characterize the functional roles of binding region components, several active‐site amino acids were mutated. A tyrosine to alanine mutated PepN (Y381A) was successfully over‐expressed, purified, and characterized. Y381A‐PepN maintained Zn(II) binding efficiency but exhibited greater than 900‐fold loss of activity (kcat) when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product. This work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Miami University.
Decline in adipogenesis contributes to peripheral subcutaneous fat loss in older humans
Authors: Caso Giuseppe , McNurlan Margaret A , Mileva Izolda , Stein Seth , Gelato Marie C
Published: June 2021

Aging is associated with redistribution of body fat characterized by a relative loss of subcutaneous limb fat and accumulation in trunk and visceral areas. The loss of subcutaneous peripheral fat is associated with metabolic abnormalities, particularly insulin resistance. The causes and mechanisms of loss of peripheral fat with aging are not completely clear.

The aim of this study was to assess whether defects in adipogenesis contribute to fat loss in aging humans, as suggested from animal studies.

Preadipocytes isolated from subcutaneous peripheral fat of healthy young and elderly subjects were compared in their ability to replicate and differentiate.

The results show that both the rate of replication of subcutaneous peripheral preadipocytes and their ability to differentiate are impaired with aging. The reduction in adipogenesis is accompanied by a higher plasma level of the inflammatory marker, soluble tumor necrosis factor receptor 2, and greater release of tumor necrosis factor α from fat tissue. Thus, the gradual loss of peripheral fat in aging humans may in part result from a defect in adipogenesis, which may be linked to inflammation and/or to increased release of proinflammatory cytokines from fat tissue.

(Supported by NIH grants DK049316 and 5‐MO1‐RR‐10710; and New York Empire Clinical Research Investigator Program (ECRIP) fellowships to GC and SS)
The binding of Vps33p to the N‐terminal domain of the yeast vacuolar syntaxin Vam3p is not required for yeast vacuole fusion
Authors: Lee Miriam , Ko Youngjun , Cho Sunglim , Jun Youngsoo
Published: June 2021

Vam3p, the syntaxin‐like yeast vacuolar Q‐SNARE, is essential for homotypic yeast vacuole fusion. Similar to other syntaxin family proteins, Vam3p possesses a three‐helical N‐terminal domain, a SNARE domain, and a C‐terminal transmembrane domain. It is generally accepted that the SNARE and transmembrane domains are required for Vam3p to mediate membrane fusion, but the requirement of the N‐terminal domain for the Vam3p function remains unclear. To examine whether the N‐terminal domain of Vam3p is required for vacuole fusion, we constructed two Vam3p constructs: one that lacks its entire N‐terminal domain and the other that contains a tobacco etch virus (TEV) protease‐cleavage site (TCS) between the N‐terminal and the transmembrane domains. The N‐terminal domain deletion either genetically or through TEV protease cleavage only moderately reduced vacuole fusion, indicating that the N‐terminal domain of Vam3p plays a marginal role in vacuole fusion. Despite this dispensability for fusion, the N‐terminal domain is strictly required for the binding of Vps33p, the Sec1/Munc18 family protein essential for vacuole fusion, to Vam3p. Thus, these results suggest that Vps33p binding to Vam3p is not required for yeast vacuole fusion.
Coordination of stress response signaling and phospholipid metabolism in yeast
Authors: Jesch Stephen A , Gaspar Maria L , Henry Susan A
Published: June 2021

Previously we showed that the availability of the lipid precursor inositol influences the levels and rates of synthesis and turnover of inositol phospholipids, phosphoinositides and complex sphingolipids. To discover the inositol‐dependent mechanisms that lead to activation of lipid‐mediated signaling pathways, we compared the simultaneous changes in lipid metabolism with those occurring in gene expression in yeast cells during a 3‐hour period of inositol starvation followed by a 30‐min period of inositol add‐back. We found that inositol starvation and add‐back led to dramatic changes in the abundance of inositol‐containing phospholipids, including phosphatidylinositol, the phosphoinositides, and complex sphingolipids. These changes were accompanied by the altered expression of hundreds of genes involved in diverse stress response pathways. Significantly we found that many mutants deleted for nonessential genes in the same stress response pathways exhibit inositol auxotrophy. These results suggest that activation of stress response signaling pathways is coordinated with inositol‐containing phospholipid metabolism.
Characterization of EHop‐016, a novel small molecule inhibitor of Rac GTPase
Authors: Humphries-Bickley Tessa Winifred , Castillo-Pichardo Linette , Montalvo-Ortiz Brenda L. , Hernandez Eliud , Vlaar Cornelis P. , Dharmawardhane Suranganie
Published: June 2021

The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs) is a viable strategy for inhibiting cancer progression. We synthesized EHop‐016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac‐GEF inhibitor NSC23766. Herein, we demonstrate that EHop‐016 inhibits Rac activity in the MDA‐MB‐435 metastatic cancer cells that overexpress Rac and exhibit high endogenous Rac activity. The IC50 of 1.1 μM for Rac inhibition by EHop‐016 is ~100‐fold lower than for NSC23766. EHop‐016 is specific for Rac1 and Rac3 at concentrations ≤ 5 μM. At higher concentrations, EHop‐016 inhibits the close homolog Cdc42. In MDA‐MB‐435 cells, EHop‐016 (≤ 5 μM) inhibits the association of the Rac‐GEF Vav2 with a nucleotide‐free Rac1(G15A), which has a high affinity for activated GEFs. However, EHop‐016 does not affect the association of the Rac‐GEF Tiam‐1 with Rac1(G15A) at similar concentrations. EHop‐016 decreases Rac‐downstream effects of p21‐activated kinase (PAK)1 activity and directed migration of metastatic cancer cells. Therefore, EHop‐016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac and Vav2 activity.
A 3D Physical Model of Karyopherin‐β2
Authors: Acheampong Mary , Ashong-Katia Rosieva , Bhikhi Kavita , Dueno Daviana , Glover Bobby , Harris Lachoy , Henry Alafia , Mata Randol , Ramirez Hillary , VanBrakle Marisa , Granberry Allison , Ketaren Natalia E. , Rout Michael P.
Published: June 2021

Karyopherin beta 2 (Kapβ2) is an importin that transports various cargo proteins into the nucleus through interactions with nucleoporins, which are proteins of the nuclear pore complex (NPC). One might overlook the significance of this protein but it actually plays a crucial role in the human body. In fact Kapβ2 is responsible for mediating transport of RNA‐binding proteins involved in RNA translation. Consequently, without this protein, we would not be able to exist. The structure of Kapβ2 is composed of 20 antiparallel helices called HEAT repeats. These HEAT repeats contribute to Kapβ2's large superhelical shape. The protein is shown to form two arches: one at the N‐terminal and the other at the C‐terminal. Through recognition of a nuclear localization signal (NLS) located on its cargo, Kapβ2 binds to its cargo via its C‐terminal arch. Release of the cargo is mediated by RanGTP, which once bound, modifies the shape of Kapβ2. This slight conformational change results in the dissociation of the substrate from its binding site. The Hostos Lincoln Academy of Science SMART (Students Modeling a Research Topic) Team and MSOE have designed and made a physical model by three‐dimensional (3‐ D) printing technology, revealing the substrate and RanGTP binding domain. Supported by grants from the HHMI Pre‐ College Program and the Camille and Henry Dreyfus Foundation.
Virtual human anatomy in medical student curriculum
Authors: Alyahya Khaleel I
Published: June 2021

It has been known that there is a tremendous amount of information with which medical professionals need to be concerned. It is increasingly apparent that human medical students need active, independent and lifelong learning skills to supplement their formal education. Advances in information and computer technology and a change toward problem‐based learning have the potential to radically change the way all medical professional students are educated. Interactive learning tools like computer‐based programs may play a significant role in enhancing this change. The computer‐based anatomy software can be incorporated into the anatomy dissection lab to address the challenges inherent in the traditional educational approach. The software uses specimen photographs and interactive features to help students study the dissection of the human body. Photographs of each phase of dissection are displayed in the software, along with dissection instructions. We propose this study that will increase our understanding of the relationships between computer technology and student achievement to determine the advantages of these software in medical field. The virtual human anatomy will be added to the curriculum of medical students to observe and detect the positive outcomes in learning human anatomy.
Mg supplementation acutely affects intracellular Mg2+ in human leukocytes
Authors: Nestler Axel , Vormann Juergen , Kolisek Martin
Published: June 2021

Intracellular Mg2+ ([Mg2+]i) is overall well regulated, however it is not known whether Mg supplementation may lead to its acute changes. Therefore we examined acute effect of Mg administered either in form of MgO or Mg citrate on total plasma Mg ([Mg]p) and [Mg2+]i in human leukocytes.

Study design

51 healthy individuals were randomized into 3 groups. One group received 300 mg Mg as MgO (n = 20), another group received 300 mg Mg as Mg‐citrate (n = 21). A control group contained 10 persons. Samples were obtained at 0 h and 1, 3, 9, and 24 h after supplementation. Probands ingested the supplement after breakfast. All probands received identical food and table water containing low‐Mg during the sampling time. [Mg]p was determined by AAS. [Mg2+]i was assessed as 340/380 nm mag‐fura 2 ratios in leukocytes of the probands.

Results

Significant changes in [Mg]p could not be observed between groups. However supplementation with Mg lead to a significant increase of 340/380 nm ratios after 3, 9, and 24 h. After 9 and 24 h, the 340/380 nm ratios were significantly higher for Mg‐citrate than for MgO.

Conclusion

Even though only minor changes in [Mg]p could be detected already 3 h after Mg supplementation [Mg2+]i increased but returned to near normal after 24 h. Mg given as Mg‐citrate induced significantly higher increases of [Mg2+]i as if given as MgO.
Proteomics reveals new evolving roles in defense and development from colostrum to mature milk
Authors: Zhang Qiang , Gao Xinliu , McMahon Robert J , Woo Jessica G , Davidson Barbara S , Morrow Ardythe L
Published: June 2021

Human milk proteins provide nutrition and support a number of vital biological processes in nursing infants. In‐depth understanding of the functions of human milk proteins has been inhibited by incomplete knowledge of the human milk proteome. We analyzed the proteomes of whey protein from human colostrum (first week; n=3) and corresponding mature milk (3 mo) using both ion‐exchange and SDS‐PAGE based protein fractionation methods. We found that the immunoglobulins sIgA and IgM are upregulated in colostrum whereas IgG is upregulated in mature milk, indicating a transformation in defense mechanism from newborns to young infants. A comprehensive view of complement components and complement regulatory proteins points to an essential role for the milk complement system prevailed by alternative pathway compared to classical pathway. A number of extracellular matrix (ECM) proteins are upregulated in colostrum. In contrast, ECM protein, fibronectin, and several actin cytoskeleton proteins it regulates are more abundant in mature milk that may underlie the important functional role of milk in regulating reactive oxygen species. The research is supported by Mead Johnson Nutrition and NIH HD 13021.